CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/185,202, filed June 26, 2015, the entire contents of which is hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under the Virginia Innovation Partnership i6 funding mechanism (Sub-Award #GG11598142515), awarded by the U.S. Department of Commerce. The government has certain rights in the invention.

FIELD

[0003] The invention relates to synthetic peptide compounds and uses thereof for therapy and diagnostics, including for complement-mediated diseases, such as inflammatory diseases, autoimmune diseases, and microbial and bacterial infections; and non-complement-mediated diseases, such cystic fibrosis and various acute diseases.

BACKGROUND

The Complement System

[0004] The complement system, an essential component of the innate immune system, plays a critical role as a defense mechanism against invading pathogens, primes adaptive immune responses, and helps remove immune complexes and apoptotic cells. Three different pathways comprise the complement system: the classical pathway, the lectin pathway and alternative pathway. Clq and mannose-binding lectin (MBL) are the structurally related recognition molecules of the classical and lectin pathways, respectively. Whereas IgM or clustered IgG serve as the principal ligands for Clq, MBL recognizes polysaccharides such as mannan. Ligand binding by Clq and MBL results in the sequential activation of C4 and C2 to form the classical and lectin pathway C3-convertase. In contrast, alternative pathway activation does not require a recognition molecule, but can amplify C3 activation initiated by the classical or lectin pathways. Activation of any of these three pathways results in the formation of inflammatory mediators (C3a and C5a) and the membrane attack complex (MAC), which causes cellular lysis. [0005] While the complement system plays a critical role in many protective immune functions, complement activation is a significant mediator of tissue damage in a wide range of autoimmune and inflammatory disease processes. (Ricklin and Lambris, "Complement- targeted therapeutics." Nat Biotechnol 2007;25(11): 1265-75).

[0006] A need exists for complement regulators. On the one hand, the complement system is a vital host defense against pathogenic organisms. On the other hand, its unchecked activation can cause devastating host cell damage. Currently, despite the known morbidity and mortality associated with complement dysregulation in many disease processes, including autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, and multiple sclerosis, only two anti-complement therapies have recently been approved for use in humans: 1) purified, human Cl-Inhibitor licensed for use in patients suffering from hereditary angioedema (HAE) and 2) eculizumab (Soliris™), a humanized, long-acting monoclonal antibody against C5 used in the treatment of paroxysmal nocturnal hemoglobinuria (P H). Both P H and HAE are orphan diseases in which very few people are afflicted. Currently, no complement regulators are approved for the more common disease processes in which dysregulated complement activation plays a pivotal role.

Dysregulated complement activation can play a role in both chronic disease indications and acute disease indications. Acute disease indications include, amongst others, acute intravascular hemolytic transfusion reaction (AIHTR), birth asphyxia, hypoxic ischemic encephalopathy, ischemia-reperfusion injury (IRI) in myocardial infarct, coronary artery bypass surgery and stroke, and solid organ transplantation rejection.

Astrovirus Coat Protein

[0007] The Astroviridae constitute a family of non-enveloped, icosahedral viruses with a single-stranded, messenger-sense RNA genome. These viruses are a significant cause of gastroenteritis in humans as well as other diseases in other animal species. It is estimated that they cause an estimated 2-17% of children's diarrheal illness worldwide.

[0008] The Astrovirus coat protein ("CP") reduces the activity of the complement system, suggesting that the 'active' portion of the protein may have clinical utility in decreasing tissue damage from complement-mediated diseases. The wild type coat protein ("WT CP") purified from human astrovirus type 1 (HAstV-1) can bind Clq and MBL, and regulates both classical and lectin pathway activation (Bonaparte et al., 2008. J. Virol. 82, 817-827; Hair et al., 2010. Molec. Immunol. 47, 792-798). This property is analogous to the properties described for human neutrophil peptide-1 (HNP-l)(Van Den Berg et al, 1998. Blood. 92, 3898-3903; Groeneveld et al, 2007. Molec. Immunol. 44, 3608-3614). The HAstV-1 coat protein is a 787 amino acid molecule that has been expressed from a recombinant baculovirus construct and then purified (Bonaparte et al, 2008. J. Virol 82, 817-827).

[0009] Developing peptide compounds to inhibit classical, lectin and alternative pathways of the complement system is needed, as each of these three pathways have been demonstrated to contribute to numerous autoimmune and inflammatory disease processes. Specific blockade of classical and lectin pathways is particularly needed, as both of these pathways have been implicated in ischemia reperfusion-induced injury in many animal models. Humans with alternative pathway deficiencies suffer severe bacterial infections. Thus, a functional alternative pathway is essential for immune surveillance against invading pathogens.

Microbial and Bacterial Infections

[0010] Many microorganisms are resistant to currently available antibiotics. Current antibiotics are derived from other microbial organisms that bacteria have competed against for space and energy over many years, which has led to rapid and predictable emergence of resistance. Some of the most pathogenic bacteria to humans are Pseudomonas aeruginosa, Staphylococcus aureus, MRSA, and carbapenemase-resistant enterobacteriacea (CREs) such as Klebsiella pneumonia. Pseudomonas aeruginosa and Staphylococcus aureus are also major pathogens in cystic fibrosis lungs. Gardnerella is a Gram variable, anaerobic coccobacillus that is a common cause of bacterial vaginosis. Gardnerella also causes complement activation and inflammation, which causes the symptoms of bacterial vaginosis and increases the risk of HIV transmission by disrupting normal barrier defenses in the vagina. There is a need for treatments for bacterial vaginosis that kill the causative organism and block inflammation. There is also a need for novel antimicrobial compounds given the increasing resistance to conventional antibiotics.

[0011] Herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) are viruses responsible for causing herpes. Infection with HSV-1 can happen from general interactions such as eating from the same utensils, sharing lip balm, or kissing. The virus is highly contagious, and it is possible to get genital herpes from HSV-1 if the individual has had cold sores and performed sexual activities during that time. Similarly, HSV-2 is also highly contagious. HSV-2 is contracted through forms of sexual contact with a person who has HSV-2. It is estimated that around 20 percent of sexually active adults within the United States have been infected with HSV-2, according to the American Academy of Dermatology (AAD). While HSV-2 infections are spread by coming into contact with a herpes sore, the AAD reports that most people get HSV-1 from an infected person who is asymptomatic, or does not have sores. Current treatments like acyclovir for HSV-1 or HSV-2 may not be fully efficacious. Thus, there is a need for anti -viral treatments for HSV-1 and HSV-2 that are more effective against viruses.

Lactobacillus Growth

[0012] Lactobacillus is a genus of bacteria that contains over 180 species. Multiple Lactobacillus species are often administered together as a single probiotic agent. In combination, various Lactobacillus species have been known to help individuals with irritable bowel syndrome, prevent necrotizing enterocolitis, and other neonatal infections. Because of its many health benefits, there is a need for compounds that can stimulate growth of Lactobacillus species.

Cystic Fibrosis

[0013] Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Patients with CF produce unusually thick, sticky mucus that clogs the lungs and leads to life-threatening lung infections, and obstructs the pancreas and stops natural enzymes from helping the body break down food and absorb vital nutrients. CF is characterized by a cycle of small airway obstruction, infection with bacterial pathogens, e.g., P. aeruginosa, Staphylococcus aureus (including methicillin- resistant S. aureus (MRSA), Burkholderia cepacia, and inflammatory lung damage.

Complement-mediated inflammation may be a major contributor to inflammatory lung damage in CF. Treatment of CF includes a regular treatment routine to maintain lung health and good nutrition. Other treatments for CF include airway clearance every day to help loosen and clear thick mucus that can build up in the lungs, inhaled medicines, including antibiotics, to help keep the airways clear, and pancreatic enzyme supplements to improve absorption of vital nutrients. There is a need for both anti-inflammatory and anti-microbial treatments for CF.

Hemolytic Transfusion Reactions

[0014] Blood transfusions can be life-saving, but can also carry the risk of a variety of reactions, some of which are potentially life-threatening, such as acute intravascular hemolytic transfusion reactions (AIHTRs). AIHTR is estimated to occur in one-fifth of total transfusions. Individuals receiving frequent blood transfusions will develop alloantibodies and autoantibodies to red blood cell (RBC) antigens over time, making cross-matching increasingly difficult and thus increasing the risk of AIHTR. Current transfusion safe-guards include "typing", "antibody screening", as well as "cross matching." While these measures have made transfusions safer, transfusion reactions still occur. AIHTR occurs when host antibodies bind to the transfused erythrocytes, initiating classical complement pathway activation, which leads to the generation of the inflammatory mediators C3a and C5a, as well as C3b opsonization and hemolysis of the transfused cells via the membrane attack complex (MAC). To date, only one case has been reported describing clinical intervention of an AIHTR by inhibiting generation of the complement anaphylatoxins C3a and C5a. No specific interventions for these reactions exist; current management of the reaction is supportive in nature. While existing safeguards make ABO incompatibility rare in the developed world, individuals with sickle cell disease and severe thalassemias requiring frequent transfusions are at increased risk for transfusion reactions due to the accumulation of antibodies against minor antigenic determinates on erythrocytes. Neonatal ABO

incompatibility in newborns can lead to jaundice, and, in severe cases, kernicterus. No blood banking organization or transfusion medicine practice has a method to directly evaluate risk for complement-mediated red blood cell lysis between donor and recipient. There is currently no effective medical intervention for ATRs except for stopping the transfusion. Diagnostic tools, prophylactic treatments to prevent AIHTR, and rescue treatments during an AIHTR are needed.

Birth Asphyxia

[0015] Birth asphyxia is a medical condition resulting from deprivation of oxygen to a newborn that causes brain damage. Hypoxic ischemic encephalopathy (HIE) is a condition that occurs when the entire brain is deprived of an adequate oxygen supply, but the deprivation is not total. HIE is most often associated with birth asphyxia. Reperfusion injury is the tissue damage caused when blood supply returns to a tissue after a period of ischemia or lack of oxygen. The absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function. Complement activation is instrumental in the development of ischemia-reperfusion injuries such as neonatal HIE. Therapeutic hypothermia (HT), the current standard of care for HIE, offers only an 11% reduction in death or disability. Published data has shown that HT paradoxically increases pro-inflammatory complement activation, which potentially limits its benefit. There is a need for treatments for HIE and birth asphyxia that regulate complement and have neuroprotective effects.

Autoimmune Hemolytic Anemia

[0016] Autoimmune hemolytic anemia (AIHA) is a disease that occurs when antibodies directed against a person's own red blood cells cause them to burst, leading to insufficient plasma concentration. AIHA is most commonly caused by IgG and IgM. IgM is a potent activator of the classical complement pathway. Thus, AIHA is characterized by complement mediated lysis of red blood cells. Thus, there is a need for therapies directed at the complement system to treat AIHA.

[0017] It would be desirable to develop peptide compounds that can regulate complement activation and can be used therapeutically to prevent and treat complement-mediated diseases, such as inflammatory and autoimmune diseases. It would also be desirable to develop peptide compounds that treat acute diseases and conditions such as acute

intravascular hemolytic transfusion reaction (AIHTR), birth asphyxia, autoimmune hemolytic anemia, viral infections, and bacterial infections, amongst others. It would also be desirable to develop peptide compounds that treat cystic fibrosis.

SUMMARY

[0018] In one aspect, the present invention provides synthetic peptide compounds that regulate the complement system and methods of using these compounds. Specifically, in some embodiments, the synthetic peptide compounds can bind, regulate and inactivate CI and MBL, and therefore can efficiently inhibit classical and lectin pathway activation at its earliest point while leaving the alternative pathway intact. These peptide compounds are of therapeutic value for selectively regulating and inhibiting CI and MBL activation without affecting the alterative pathway and can be used for treating diseases mediated by

dysregulated activation of the classical and lectin pathways. In other embodiments, the peptide compounds regulate classical pathway activation but not lectin pathway activation. The peptide compounds are useful for various therapeutic indications - even for indications that are unrelated to complement regulation. In some embodiments, these peptide compounds are of therapeutic value for treating acute diseases and conditions such as acute intravascular hemolytic transfusion reaction (AIHTR), birth asphyxia, as well as bacterial, viral, and antimicrobial infections, amongst others. These peptide compounds are also of therapeutic value for treating cystic fibrosis. [0019] In some embodiments, the invention is based on the identification and modification of peptides of 15 amino acids from Polar Assortant (PA) peptide (SEQ ID NO: 3), derivatives of the peptides, and methods of their use. The PA peptide is a scrambled peptide derived from human astrovirus protein, called CPl (SEQ ID NO: 1). The PA peptide is also known as PICl (Peptide Inhibitors of Complement CI), AstroFend, AF, or SEQ ID NO: 3. As used herein, the term "PICl peptides" include SEQ ID NO: 3 as well as other amino acid sequences that are the same as SEQ ID NO:3 but with PEGylation modifications. The PICl peptide was originally named as such because it was found to be associated with diseases mediated by the complement system. In some aspects, surprisingly, the invention is also related to use of the PA peptide and derivatives for treating diseases and conditions that are not associated with the complement system, such as, but not limited to, cystic fibrosis and chronic obstructive pulmonary disease (COPD). In some embodiments, the invention is based on the PICl peptide and modifications thereof, having the amino acid sequences and modifications as set forth in SEQ ID NOs: 3-47. These peptides can be used to regulate complement activation, including complement inhibition; treat and/or prevent hemolytic reactions; treat hypoxic ischemic encephalopathy; treat cystic fibrosis; for anti-microbial use; and to treat and/or prevent other diseases and conditions disclosed herein.

[0020] In some aspects, the invention is directed to peptide compounds that are peptide mimetics, peptide analogs and/or synthetic derivatives of PA having, for example, internal peptide deletions and substitutions, deletions and substitutions at the N terminus and C terminus, and that are able to regulate the classical and lectin pathway activation by binding to Clq and MBL.

[0021] A further embodiment of the invention is any one of the peptide compounds of this invention, wherein the peptide compound is modified through sarcosine substitution, alanine substitution, and/or PEGylation of the N terminus, C terminus, or N terminus and C terminus.

[0022] In some embodiments, the peptide sequence has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%), at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 3-47.

[0023] In one aspect, the invention provides a method of treating and/or preventing a hemolytic reaction in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47. In other embodiments, the hemolytic reaction is selected from the group consisting of acute intravascular hemolytic transfusion reaction (AIHTR), transfusion related acute lung injury (TRALI), and platelet transfusion refractoriness. In further embodiments, the composition is administered before the subject is administered a blood transfusion, after the subject is administered the blood transfusion, and/or during the blood transfusion.

[0024] In another aspect, the invention provides a method of treating hypoxic ischemic encephalopathy in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3- 47.

[0025] In another aspect, the invention provides a method of treating cystic fibrosis in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47.

[0026] In another aspect, the invention provides a method of treating a bacterial infection in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the bacterial infection is caused by bacteria selected from the group consisting of Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, and Gardnerella sp. In further

embodiments, the bacterial infection is caused by Gram-positive or Gram-negative bacteria.

[0027] In another aspect, the invention provides a method of enhancing Lactobacillus growth in a subject comprising: administering to the subject a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47.

[0028] In another aspect, the invention provides a method of treating a viral infection in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the viral infection is caused by herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2).

[0029] In another aspect, the invention provides a method of treating autoimmune hemolytic anemia in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47. The autoimmune hemolytic anemia may be characterized by one or more of elevated serum bilirubin, excess urinary urobilinogen, reduced plasma haptoglobin, raised serum lactic dehydrogenase (LDH), hemosiderinuria, methemalbuminemia, spherocytosis, reticulocytosis, and/or erythroid hyperplasia of the bone marrow.

[0030] In another aspect, the invention provides a method of treating birth asphyxia in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47. In some

embodiments, presence of birth asphyxia is characterized by an Apgar score of 3 or under that lasts five minutes or more.

[0031] In another aspect, the invention provides a method of treating acute kidney injury in a subject in need thereof comprising: administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3-47. In some embodiments, the synthetic peptide has the amino acid sequence and modifications selected from the group consisting of SEQ ID NO: 3-47.

[0032] In another aspect, the invention is a synthetic peptide comprising at least about 90% sequence identity to an amino acid sequence of SEQ ID NO: 3-47. In some

embodiments, a pharmaceutical composition can comprise a therapeutically effective amount of the synthetic peptide and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0033] In another aspect, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NO: 3-47. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NO: 4- 18 and 30-47. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NO: 3 and 19-29. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and PEGylation modifications of SEQ ID NO: 21. In some embodiments, a pharmaceutical composition can comprise a therapeutically effective amount of the synthetic peptide and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0034] In another aspect, the invention is directed to a PICl peptide, wherein the peptide is modified through PEGylation of the N terminus, the C terminus, or both the N terminus and C terminus. In another aspect, the invention is directed to a PICl peptide, wherein the peptide has been modified by sarcosine and/or alanine substitutions. In other embodiments, the peptide is modified through PEGylation and sarcosine and/or alanine substitutions. In some embodiments, the peptides are isolated and/or purified.

[0035] In one aspect, the invention provides a method of selecting at least one peptide for treating a subject having cystic fibrosis comprising: (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity on cystic fibrosis; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity on cystic fibrosis.

[0036] In another aspect, the invention provides a method of selecting at least one peptide for treating a subject having a hemolytic reaction comprising: (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity on the hemolytic reaction; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity on the hemolytic reaction. In some embodiments, the hemolytic reaction is selected from the group consisting of AIHTR, transfusion related acute lung injury (TRALI), and platelet transfusion refractoriness.

[0037] In another aspect, the invention provides a method of selecting at least one peptide for treating a subject having a bacterial infection comprising: (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity on the bacterial infection; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity on the bacterial infection. In some embodiments, the bacterial infection is caused by bacteria selected from the group consisting of Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, and Gardnerella sp. In some embodiments, the bacterial infection is caused by Gram-positive or Gram-negative bacteria.

[0038] In another aspect, the invention provides a method of selecting at least one peptide for enhancing growth of Lactobacillus comprising (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity enhancing growth of Lactobacillus species; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity enhancing growth of Lactobacillus species.

[0039] In another aspect, the invention provides a method of selecting at least one peptide for treating and/or preventing a viral infection comprising (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity that treats or prevents the viral infection; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity that treats or prevents the viral infection. In some embodiments, the viral infection is caused by HSV-1 or HSV-2.

[0040] In another aspect, the invention provides a method of selecting at least one peptide for treating and/or preventing autoimmune hemolytic anemia comprising (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity on autoimmune hemolytic anemia; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity on autoimmune hemolytic anemia.

[0041] In another aspect, the invention provides a method or selecting at least one peptide for treating birth asphyxia comprising (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity on birth asphyxia; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity on birth asphyxia. [0042] In another aspect, the invention provides a method of selecting at least one peptide for treating hypoxic ischemic encephalopathy comprising: (a) testing peptides selected from the group consisting of SEQ ID NOs: 3-47 for activity on hypoxic ischemic encephalopathy; and (b) selecting from the group consisting of SEQ ID NOs: 3-47 at least one synthetic peptide having activity on hypoxic ischemic encephalopathy.

[0043] In other aspects, the invention provides a method of treating a disease by administering the compositions described herein, wherein the disease that is at least partially complement mediated includes but is not limited to: hemolytic transfusion reactions, cold- agglutinin disease, immune-complex diseases, thalassemia, sickle cell disease, ABO incompatibility, acute/hyperacute solid organ transplantation rejection, instant blood- mediated inflammatory reaction (IBMIR), solid organ transplantation warm/cold ischemia, systemic lupus erythematosus (SLE), rheumatoid arthritis, ischemia-reperfusion injury, myocardial infarct, stroke, hypoxic ischemic encephalopathy, traumatic brain injury, coronary artery bypass surgery, wound healing, cancer, Alzheimer's disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, Crohn's disease, Sepsis syndrome/ARDS/SIRS, glomerulonephritis, lupus nephritis, anti -glomerular basement membrane disease, anti-neutrophil cytoplasmic autoantibody-induced, membranoproliferative glomerulonephritis, dense deposit disease, membranous nephropathy, IgA nephropathy, or C3 glomerulopathy.

[0044] In another aspect, the invention provides a method of treating a disease by administering the compositions described herein, wherein the disease is not complement- mediated, which includes but is not limited to cystic fibrosis and chronic obstructive pulmonary disease (COPD).

[0045] Another embodiment of the invention is a method of treating a disease associated with complement-mediated tissue damage, further comprising administering to a subject at least one other active ingredient effective in treating the disease, wherein at least one other active ingredient includes a non-steroidal anti-inflammatory agent, a corticosteroid, a disease modifying anti-rheumatic drug, CI -inhibitor, and eculizumab.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0047] Other features and advantages of the invention will be apparent from the detailed description, drawings, and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0048] FIG. 1 shows the titration of PA-dPEG24 (SEQ ID NO: 21), PA-P7Sar (SEQ ID NO: 10), and PA-C9Sar (SEQ ID NO: 12), in a hemolytic assay using factor B depleted serum. Concentrations of peptides are shown in mM.

[0049] FIGS. 2 A-B show THAT P A-dPEG24 (SEQ ID NO : 21 ) inhibits complement activation in a hemolytic assay using factor B depleted serum to the same degree as PA (SEQ ID NO: 3).

[0050] FIG. 3 shows the structure of PA-dPEG24 (SEQ ID NO: 21).

[0051] FIGS. 4A-E show plate dilution assays. FIG. 4A shows plate dilution assays for Staphylococcus aureus treated with PA-dPEG24 (SEQ ID NO: 21) or saline control. The X- axis shows inhibitor concentration of PA-dPEG24 (SEQ ID NO: 21) in mg/ml. FIG. 4B shows plate dilution assays for Staphylococcus aureus treated with PA-dPEG24 or

Vancomycin. The X-axis shows inhibitor concentration of PA-dPEG24 (SEQ ID NO: 21) in mg/ml, and vancomycin starting at 5 μg/ml. FIG. 4C shows plate dilution assays for Klebsiella pneumonia treated with PA-dPEG24 (SEQ ID NO: 21) or Gentamycin. The X- axis shows inhibitor concentration of PA-dPEG24 (SEQ ID NO: 21) in mg/ml. Gentamycin starting at 4μg/ml. FIG. 4D shows plate dilution assays for Pseudomonas aeruginosa treated with PA-dPEG24 (SEQ ID NO: 21) or Gentamycin control. The X-axis shows inhibitor concentration of PA-dPEG24 (SEQ ID NO: 21) in mg/ml. Gentamycin starting at 4 μg/ml. FIG. 4E shows plate dilution assays for Pseudomonas aeruginosa treated with four different versions of PA-dPEG24 (SEQ ID NO: 21) or Gentamycin control. Figure Legend: PICl = PA-dPEG24 (SEQ ID NO: 21); PA-L3Sar (H2N-IA(Sar)ILEPICCQERAA-OH) (SEQ ID NO: 6); PA-I4Sar (H2N-IAL(Sar)LEPICCQERAA-OH) (SEQ ID NO: 7); PA-L5Sar (H2N- IALI(Sar)EPICCQERAA-OH) (SEQ ID NO: 8).

[0052] FIG. 5 shows inhibition of Gardnerella growth in the presence of PA-dPEG24 (SEQ ID NO: 21), PA-L3Sar (SEQ ID NO: 6), PA-I4Sar (SEQ ID NO: 7), or PA-L5Sar (SEQ ID NO: 8). PICl = PA-dPEG24 (SEQ ID NO: 21); Sarc-1 = PA-L3Sar (SEQ ID NO: 6); Sarc-2 = PA-I4Sar (SEQ ID NO: 7); Sarc-3 = PA-L5Sar (SEQ ID NO: 8).

[0053] FIG. 6 shows C5a release when serum is incubated with Gardnerella at different concentrations of PA-dPEG24 (SEQ ID NO: 21). PICl = PA-dPEG24 (SEQ ID NO: 21).

[0054] FIG. 7 shows two type-0 plasmas from the RBC transfusions that caused ATR in a blood type-B recipient. The IgG titers for each plasma was quite high, but non- discriminatory. However, in a CH50-type hemolytic assay, they behave drastically differently. One is highly hemolytic, while the other does not cause significant hemolysis.

[0055] FIG. 8 shows the type-0 plasma (#426) from the RBC transfusions that caused ATR in blood type-B recipient. Prior to adding B erythrocytes, PA-dPEG24 (SEQ ID NO: 21) was added to the plasma in increasing concentrations. A dose-response inhibition of hemolysis was demonstrated with PA-dPEG24 (SEQ ID NO: 21) demonstrating up to >95% inhibition.

[0056] FIG. 9 shows bacteria initiating classical pathway complement activation with C5a-mediated neutrophil recruitment and activation.

[0057] FIGS. 10A-D shows complement anaphylatoxins in CF and control lung fluid. FIG. 10A: C5a concentrations in soluble (sol) fractions from sputum of CF patients (n=15)

th th

and controls (n=3). Box shows quartiles, whiskers are 90 and 10 percentile, and dashed line is the mean. CF sols C5a level is 5-fold higher than control sols (P = 0.04). FIG. 10B: C5a Western blot for sputum sols for two healthy controls (A and B) and 2 CF subjects (X and Y). FIG. IOC: C3a concentrations in sol fractions from sputum of CF patients (n=14)

th ^th

and controls (n=4). Box shows quartiles, whiskers are 90 and 10 percentile, and dashed line is the mean. P = 0.03. FIG. 10D: C4a concentrations in sol fractions from sputum of CF

th th

patients (n=15) and controls (n=5). Box shows quartiles, whiskers are 90 and 10 percentile, and dashed line is the mean. P = 0.05.

[0058] FIGS. 11A-B shows complement opsonization of Staphylococcus aureus. FIG. 11A: Staphylococcus aureus-bound C3-fragments after incubation in sol fractions from

th sputum of CF patients (n=5) and controls (n=3). Box shows quartiles, whiskers are 90 percentile, and dashed line is the mean. P = 0.42. FIG. 11B: Staphylococcus aureus-bound C4-fragments after incubation in sol fractions from sputum of CF patients (n=5) and controls

th th

(n=3). Box shows quartiles, whiskers are 90 and 10 percentile, and dashed line is the mean. P = 0.13.

[0059] FIGS. 12A-D shows C5a generated by bacteria in CF sols. FIG. 12A: C5a concentrations in sol fractions from sputum of CF patients (n=3) or controls (n=3) before and after incubation with live or dead Pseudomonas aeruginosa or Staphylococcus aureus. Data are means ± SE. C5a was generated in CF sol in the presence of Pseudomonas aeruginosa (P = 0.03) or Staphylococcus aureus (P = 0.03). FIG. 12B: C5a concentrations in sol fractions from sputum of CF patients (subjects A, B, and C) before (Initial) and after incubation with live or dead Pseudomonas aeruginosa. FIG. 12C: C5a concentrations in sol fractions from sputum of CF patients (subjects A, B, and C) before (Initial) and after incubation with live or dead Staphylococcus aureus. FIG. 12D: C5a concentrations in CF sols that were incubated alone in buffer (CF sol only), incubated with dead Pseudomonas aeruginosa (CF sol + Pseudomonas aeruginosa), or incubated with PA-dPEG24 (SEQ ID NO: 21) and dead Pseudomonas aeruginosa (CF sol + PA-dPEG24 + P. aerug). PA-dPEG24, SEQ ID NO: 21 reduces C5a generation in CF sols incubated with Pseudomonas aeruginosa.

[0060] FIGS. 13A-C shows correlation plots for complement effectors and clinical measures. FIG. 13A: C5a concentrations in CF sols positively correlate with increasing age, r = 0.53, P = 0.04. FIG. 13B: C5a concentrations in CF sols correlate inversely with BMI percentile in children, r = -0.77, P = 0.04. FIG. 13C: C3a concentrations in CF sols positively correlate with FEV1%, r _s = 0.63, P = 0.02.

[0061] FIGS. 14A-E shows peptide compounds binding and inhibiting Clq. FIG. 14A: shows PA (SEQ ID NO: 3) binding to Clq, but not to CRT or BSA. FIG. 14B shows PICl

(SEQ ID NO: 21) inhibits DyLight 680 labeled Clq binding in recombinant CRT. FIG. 14C shows PICl (SEQ ID NO: 21) inhibits DyLight labeled 488 Clq binding to Raji cells, and

FIG. 14D shows representative In-Cell Western plate assay imaged on LICOR Odyssey demonstrating that increasing amounts of PICl (SEQ ID NO: 21) inhibit DyLight 680 labeled

Clq from binding Raji cells. FIG. 14E shows the experiment setup.

[0062] FIG. 15 shows soluble Clq binding calreticulin (CRT), but not BSA.

[0063] FIG. 16 shows PA (SEQ ID NO: 3) inhibiting Clq binding to calreticulin (CRT) compared to a negative control peptide CP2.

[0064] FIGS. 17A-C shows Raji cells at 20x magnification. FIG. 17B: FITC (Clq) at l/50s exposure. FIG. 17C: DAPI at l/500s exposure. FIG. 17A: Overlay.

[0065] FIGS. 18A-C shows Raji cells at 20x magnification treated with 0.523mM PA- dPEG24 (SEQ ID NO: 21). FIG. 18B: FITC (Clq) at l/50s exposure. FIG. 18C: DAPI at l/500s exposure. FIG. 18A: Overlay.

[0066] FIGS. 19A-C shows Raji cells at 20x magnification treated with 1.05mM PA- dPEG24 (SEQ ID NO: 21). FIG. 19B: FITC (Clq) at l/50s exposure. FIG. 19C: DAPI at l/500s exposure. FIG. 19A: Overlay.

[0067] FIG. 20 shows red blood cell lysis in a hemolytic assay in rats treated with two different doses of PA-dPEG24 (SEQ ID NO: 21) or with normal saline. [0068] FIGS. 21A-B shows the experimental design and study arms. FIG. 21A shows PA-dPEG24 (SEQ ID NO: 21) dosing in AIHTR model, FIG 21B shows PA-dPEG24 (SEQ ID NO: 21) efficacy in AIHTR model.

[0069] FIGS. 22A-B shows PA-dPEG24 (SEQ ID NO: 21) dosing studies in AIHTR model comparing high dose PA-dPEG24 (SEQ ID NO: 21) (40mg) vs. lose dose PA-dPEG24 (SEQ ID NO: 21) (20mg). FIG. 22A shows free hemoglobin present in rat plasma collected at 0 sec (prebleed), 30seconds, 5 minutes, 20 minutes, 60 minutes, and 120 minutes after 15% transfusion of human red blood cells (HuRBCs), measured by spectrophotometry. Vehicle control is saline. Positive control is cobra venom factor (CVF). n= number of animals in each group. Error bars denote standard error of the means. FIG. 22B shows percent of surviving HuRBCs (detected using FITC-conjugated anti -human CD235a (glycophorin A monoclonal antibody) at 30 seconds, 5 minutes and 20 minutes after transfusion measured by flow cytometry. Error bars denote standard error of the means. CVF: cobra venom factor. Saline: 0.9% normal saline.

[0070] FIGS. 23A-C shows PA-dPEG24 (SEQ ID NO: 21) efficacy in prophylaxis (pre- transfusion) vs. treatment (rescue therapy, given after transfusion) in AIHTR model. FIG. 23A shows free hemoglobin present in rat plasma collected at 0 second (pre-bleed), 30 seconds, 5 minutes, 20 minutes, 60 minutes, and 120 minutes after 15% transfusion of HuRBCs, measured by spectrophotometry. Error bars denote standard error of the means. FIG. 23B shows area under the curve (total hemoglobin released) over 120 minutes. Error bars denote standard error of the means. FIG. 23C shows unconjugated bilirubin measured at 120 minutes compared to pre-transfusion (pre-bleed). Error bars denote standard error of the means.

[0071] FIGS. 24A-D shows PA-dPEG24 (SEQ ID NO: 21) protection of human RBCs from lysis by rat serum in vitro. FIG. 24A shows flow cytometry analysis of HuRBCs incubated for 5 min in rat serum then labeled with anti-glycophorin A (APC) and anti-C3 (FITC). Labeled opsonized RBCs spiked into unlabeled RBCs (representative plot). FIG. 24B shows flow cytometry analysis of HuRBCs incubated 5 min in rat serum treated with PA-dPEG24 (representative plot). FIG. 24C shows relative increase in number of HuRBCs with no C3 deposition (Q3) in PA-dPEG24 treated serum compared to untreated serum after 5 min. Error bars denote standard error of the means for two independent experiments. FIG. 24D shows percent of serum-incubated RBCs that bound C3-fragments (Q2/Q2+Q3) is reduced in PA-dPEG24 treated serum compared to untreated serum after 5 min. Error bars denote standard error of the means for 2 independent experiments. Q2: Cells dual stained with C3 and glycophorin A (C3 deposition on glycophorin A labeled cells). Q3 : Cells with glycophorin A label (intact cells).

[0072] FIGS. 25A-F shows PA-dPEG24 (SEQ ID NO: 21) protection versus saline of human RBCs transfused into rats at 30 seconds (FIGS. 25A and 25D), 5 minutes (FIGS. 25B and 25E) and 20 minutes (FIGS. 25C and 25F). Flow cytometry analysis of RBCs recovered from blood draws and labeled with anti-glycophorin A (APC) and anti-C3 (FITC). In FIGS 25A-C, the animal was given saline control. In FIGS. 25D-F, the animal was prophylactically treated with PA-dPEG24 (SEQ ID NO: 21). Q2: Cells dual stained with C3 and glycophorin A. Q3 : Cells with glycophorin A label (intact HuRBCs).

[0073] FIGS. 26A-D show transfused human RBC survival in rats. FIG. 26A shows the number of events measured at 30 seconds after transfusion with HuRBCs showing all anti gpA+ RBCs (Q2+Q3), anti gpA+ anti C3- (Q3) and anti gpA+ anti C3+ (Q2) for PA-dPEG24 (SEQ ID NO: 21) prophylaxis, saline control, CVF (cobra venom factor) control and PA- dPEG24 (rescue) treatment. Error bars denote standard error of the means. FIG. 26B shows percent of anti gpA+, anti C3+ RBCs compared with total anti gpA+ RBCs (Q2/ Q2+Q3). Error bars denote standard error of the means. FIG. 26C shows total anti gpA + RBCs (Q2+Q3) present in blood at 5 minutes and 20 minutes compared to the controls (Saline and CVF). Error bars denote standard error of the means. FIG. 26D shows anti gpA- anti C3+ RBCs (Ql) present in blood up to 20 minutes. Legend: PICl (SEQ ID NO: 21) prophylaxis is denoted with a star; PICl (SEQ ID NO: 21) Treatment (rescue treatment compared with saline control) is denoted with a diamond; saline control is denoted with a square; CVF control is denoted with a triangle. Error bars denote standard error of the means.

[0074] FIGS. 27A-F shows PA-dPEG24 (SEQ ID NO: 21) protection of human RBCs transfused into rats as compared to immune globulin intravenous (IVIG) at 30 seconds

(FIGS. 27A and 27D), 5 minutes (FIGS. 27B and 27E) and 20 minutes (FIGS. 27C and 27F) post-transfusion. Flow cytometry analysis of RBCs recovered from blood draws and labeled with anti-glycophorin A (APC) and anti-C3 (FITC). FIGS. 27A-C show the animal prophylactically treated with IVIG. FIGS. 27D-F show the animal prophylactically treated with PA-dPEG24. Q2 shows cells dual stained with C3 and glycophorin A. Q3 shows cells with glycophorin A label (intact HuRBCs).

[0075] FIGS. 28A-C shows prophylactic PA-dPEG24 (SEQ ID NO: 21) efficacy as compared to prophylactic immune globulin intravenous (IVIG). FIGS. 28A shows free hemoglobin present in rat plasma collected at 0 second (pre-bleed), 30 seconds, 5 minutes, 20 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes after 15% transfusion of HuRBCs, measured by spectrophotometry. Error bars denote standard error of the means. FIG. 28B shows number of events measured at 30 seconds after transfusion with HuRBCs showing all anti gpA+ RBCs (Q2+Q3), anti gpA+ anti C3- (Q3) and anti gpA+ anti C3+ (Q2) for PA-dPEG24 prophylaxis and IVIG prophylaxis. Error bars denote standard error of the means. FIG. 28C shows anti gpA- anti C3+ RBCs (Ql) present in blood at 30 seconds for PA-dPEG24 prophylaxis and IVIG prophylaxis. Error bars denote standard error of the means. FIG. 28D shows Total anti-gpAl RBCs for IVIG (no lines) and PICl (diagonal lines) prophylaxis at 5 and 20 minutes after transfusion.

[0076] FIGS. 29A-C shows prophylactic PA-dPEG24 (SEQ ID NO: 21)(labeled as PICl) efficacy as compared to prophylactic immune globulin intravenous (IVIG) on acute kidney injury. FIG. 29A shows gross kidney weights measured for IVIG prophylaxis and PA-dPEG24 prophylaxis prior to formalin fixing. Error bars denote standard error of the means, n=6. FIG. 29B shows gross kidney image for IVIG prophylaxis and PA-dPEG24 prophylaxis prior to formalin fixing (representative animal). FIG. 29C Representative histology (hematoxylin and eosin stain) of kidneys from rats receiving saline (i, ii), IVIG prophylaxis (iii, iv), or PICl prophylaxis (v, vi). PICl -treated rats demonstrate normal kidney architecture, whereas saline- and IVIG-treated rats show disruption of cellular architecture consistent with acute tubular necrosis. Bar represents 20 mm. Tissues were observed with a microscope (Bmax, Olympus) at a magnification of 4003 at room temperature. Images were acquired with a digital camera (DP71, Olympus).

[0077] FIG. 30 shows percent maximal serum hemolysis after hypoxia in rats at 1, 2, 4, 8, 16, 24, 48 hours post-treatment with PICl (PA-dPEG24) (SEQ ID NO: 21), cobra venom factor (CVF), or therapeutic hypothermia (31-32°C for 6 hours).

[0078] FIG. 31 shows images of brain slices after hypoxia in rats treated with PICl peptide (PA-dPEG24), cobra venom factor (CVF) therapeutic hypothermia (31-32°C for 6 hours).

[0079] FIG. 32A shows combined Clq cranial levels in HIE. There is significantly less Clq deposition in the PICl (SEQ ID NO: 21) treated group (Normothermia + PICl (SEQ ID NO: 21) - triangle) when compared to untreated HIE controls (Normothermia - diamond) at 4, 12, and 24 hours after brain injury. PICl (SEQ ID NO: 21) treated animals had similar levels of Clq compared to animals who underwent therapeutic hypothermia (square). [0080] FIG. 32B shows evidence of neuroprotection post-HIE with PICl (SEQ ID NO: 21) treatment. Panels A-D of FIG. 32B show cresyl violet staining. Cresyl violet stains Nissl substance in neurons. The HIE group (Panel B) shows a significant decrease in cresyl violet staining compared to the PICl (SEQ ID NO: 21) group (Panel D), indicating the neuro- preservatory action of PICl (SEQ ID NO: 21) similar to therapeutic hypothermia (Panel C). Panels E-H show hematoxylin and eosin staining. Hematoxylin and Eosin staining demonstrated a greater degree of neuronal destruction (necrosis, pyknosis and karyorrhexis) in the HIE brain (Panel F) when compared to those treated with PICl (SEQ ID NO: 21) (Panel H). Panels I-L show acridine orange staining. Acridine orange stains viable parts of the brain a bright green (Panels K and L). HIE significantly decreases acridine orange staining in the brain (Panel J). PICl (SEQ ID NO: 21) treatment restored acridine orange staining, indicating the presence of more viable cells in the brain (Panel L).

[0081] FIG. 32C shows histological neuroprotection also translated into neurofunctional improvement after HIE. Animals injected with PICl (SEQ ID NO: 21) performed better than normothermic animals and similar to no-intervention animals or animals receiving therapeutic hypothermia (No intervention, first bar on the left; HIE: Normothermia, second bar from left; HIE+therapeutic hypothermia: Hypothermia, third bar from left,

HIE+PIC1(SEQ ID NO: 21): Normo-PICl (SEQ ID NO: 21), fourth bar from left).

[0082] FIG. 33 shows PICl (SEQ ID NO: 21) vs. Acyclovir inhibition of HSV-1 encoding GFP. In the no virus, negative control (as labeled), the histogram shows a peak to the left, indicating that there are no infected cells (GFP-). In the positive control, the peak shifts to the right, indicating infected cells (GFP+). In row 2, an increasing concentration of PICl (SEQ ID NO: 21) gradually shifts the peak to the left, indicating that PICl (SEQ ID NO: 21) inhibits viral replication. In contrast, row 3 shows the dose-dependent effect of Acyclovir (ACV) on viral replication. Treatment with 5mM of ACV resulted in a small right peak, indicating that there was still residual viral replication.

[0083] FIG. 34 shows PICl (SEQ ID NO: 21) vs. Acyclovir inhibition of HSV-2. In the negative control (as labeled), the histogram shows a peak to the left, indicating that there are no infected cells (GFP-). In the positive control, the peak shifts to the right, indicating infected cells (GFP+). In row 2, an increasing concentration of PICl (SEQ ID NO: 21) gradually shifts the peak to the left, indicating that PICl (SEQ ID NO: 21) inhibits viral replication. In contrast, row 3 shows the dose-dependent effect of Acyclovir (ACV) on viral replication. [0084] FIG. 35 shows that PICl (SEQ ID NO: 21), termed AF1 on the x-axis of the figure, promotes the growth of L. acidophilus and L. leichmannii.

[0085] FIG. 36A-C shows the antimicrobial activity of PICl (SEQ ID NOS: 7, 8, 12, and 21). FIG. 36A shows that PICl (SEQ ID NO: 21) inhibits growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae in microdilution MIC testing. FIG. 36B shows peptide variants AF1-AF5 (SEQ ID NO: 21, 5, 6, 7 and 8, respectively) in microdilution MIC testing for Pseudomonas aeruginosa. FIG. 36C shows confocal microscopy showing PICl (SEQ ID NO: 21) bound to the outer surface of bacteria.

[0086] FIG. 37A-D shows Neisseria gonorrhoeae growth in presence or absence of PICl (SEQ ID NO: 21). FIG. 37A shows Neisseria gonorrhoeae growth in presence or absence of 20mg/ml of PICl (SEQ ID NO: 21). FIG. 37B shows Neisseria gonorrhoeae growth in presence or absence of 30mg/ml of PICl (SEQ ID NO: 21). FIG. 37C shows Neisseria gonorrhoeae grown in titration concentrations of PICl (SEQ ID NO: 21). FIG. 37D shows colony counts from Neisseria gonorrhoeae incubated with and without PICl (SEQ ID NO: 21).

[0087] FIG. 38 shows PICl (SEQ ID NO: 21) inhibits MPO activity in sputum samples (sol) isolated from cystic fibrosis (CF) patients. CF sol samples were incubated in the presence or absence of 20 mg/ml PICl (SEQ ID NO: 21) for 30 minutes followed by addition of TMB for 30 minutes at room temperature. MPO activity was then measured by detection of TMB color change in a spectrophotometer at 450 nm.

[0088] FIG. 39 shows PICl (SEQ ID NO: 21) dose-dependently inhibits MPO activity in a sputum samples (sol) isolated from a cystic fibrosis (CF) patient. The CF sol was incubated in the presence of increasing amounts of PICl (SEQ ID NO: 21) at room temperature. MPO activity was then measured by detection of TMB color change in a spectrophotometer at 450 nm.

[0089] FIG. 40 shows PICl (SEQ ID NO: 21) inhibits MPO activity in rat brains subject to hypoxic ischemic encephalopathy (HIE). Supernatants from rat brain lysate that underwent HIE was incubated in the presence or absence of 20 mg/ml PICl (SEQ ID NO: 21). MPO activity was then measured by detection of TMB color change in a spectrophotometer at 450 nm.

[0090] FIG. 41 shows brain lysates from rat pups with HIE either receiving no treatment (NT = normothermia), Hypothermia (HT), or PICl (SEQ ID NO: 21) i.p. (NP =

normothermia + PICl (SEQ ID NO: 21)). The lysates were then tested for MPO activity with TNB. Over 48 hours the NT animals experience increasing MPO activity consistent with increased reperfusion injury and infarction size. The PICl (SEQ ID NO: 21) treated animal shows a trend towards decreased MPO activity compared with no treatment (NT) at each time point, suggesting a decrease in MPO activity in the HIE brain after i.p. administration of PICl (SEQ ID NO: 21).

[0091] FIG. 42 shows that PICl (SEQ ID NO: 21) dose-dependently inhibits MPO activity in lysates of purified human neutrophils (PMN). PMN lysates were incubated in the presence of increasing amounts of PICl (SEQ ID NO: 21). MPO activity was then measured by detection of TMB color change in a spectrophotometer at 450 nm.

[0092] FIG. 43 shows titration of the inhibition of MPO activity by increasing amounts of PICl (SEQ ID NO: 21). PICl (SEQ ID NO: 21) directly inhibits MPO.

[0093] FIG. 44 shows that increasing amounts of RBC lysates containing hemoglobin demonstrated a dose-dependent increase in the oxidation of the chromogen

tetramethylbenzidine (TMB) substrate. In the presence of PICl (SEQ ID NO: 21) at

20mg/ml, the oxidation of TMB was inhibited.

[0094] FIG. 45 shows increasing amounts of PICl (SEQ ID NO: 21) led to a dose- dependent decrease in oxidized TMB signal by hemoglobin from RBC lysates.

[0095] FIG. 46 shows that increasing amounts of PICl (SEQ ID NO: 21) dose- dependently inhibited oxidation of tetramethylbenzidine TMB with increasing amounts of hemoglobin from RBC lysate.

DETAILED DESCRIPTION

[0096] The present invention provides synthetic peptide compounds based on the modification of isolated, purified peptides of 15 amino acids from Polar Assortant (PA) peptide (SEQ ID NO: 3), derivatives of the peptides, and methods of their use. The PA peptide is a scrambled, shortened peptide derived from human astrovirus protein, called CP1 (SEQ ID NO: 1). In some embodiments, the invention is based on modification of an isolated, purified peptide of CP1, and the peptide derivatives have the amino acid sequences set forth in SEQ ID NOs:4-47. SEQ ID NOs: 4-47 are derivatives of PA (SEQ ID NO: 3) having, for example, sarcosine substitutions and/or modifications at the N terminus and C terminus, including PEGylation. These peptides can be used to regulate complement activation, including complement inhibition; treat and/or prevent hemolytic reactions; treat hypoxic ischemic encephalopathy; treat cystic fibrosis; for anti-microbial use; and treat and/or prevent other diseases and conditions disclosed herein.

[0097] In some aspects, these peptide compounds are of therapeutic value for the treatment of diseases and conditions mediated by dysregulated activation of the classical and lectin pathways. In some aspects, the invention provides methods for selecting peptides that have anti-microbial activity, can be used to treat cystic fibrosis, and/or can be used to treat various acute diseases. The PA peptide (SEQ ID NO: 3) has low solubility in aqueous solutions. The peptides of SEQ ID NO: 21 and other PEGylated and sarcosine substitutions (e.g., SEQ ID NOs: 5-8, 10-12, 21-25, 27-29, 30-40, 43, 45, and 47), have increased solubility in aqueous solutions, which may increase their efficacy as therapeutic compounds.

[0098] In some embodiments, the invention is based on the identification and

modification of an isolated, purified peptide of 30 amino acids derived from human astrovirus coat protein, termed CP1, and having a sequence (SEQ ID NO: 1) that is able to regulate the classical and lectin pathway activation by binding to Clq and MBL. In other embodiments, the peptide compounds regulate the classical pathway activation but not the lectin pathway activation.

[0099] Modifications of the amino acid structure of CP1 has led to the discovery of additional peptide compounds that are able to regulate complement activation, such as Clq activity.

[0100] The term "peptide compound(s)," as used herein, refers to amino acid sequences, which may be naturally occurring, or peptide mimetics, peptide analogs and/or synthetic derivatives of about 15 amino acids based on SEQ ID NO: 3. In addition, the peptide compound may be less than about 15 amino acid residues, such as between about 10 and about 15 amino acid residues and such as peptide compounds between about 5 to about 10 amino acid residues. Peptide residues of, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 amino acids are equally likely to be peptide compounds within the context of the present invention. Peptide compounds can also be more than 15 amino acids, such as, for example, 16, 17, 18, 19, and 20, or more amino acids.

[0101] The disclosed peptide compounds are generally constrained (that is, have some element of structure as, for example, the presence of amino acids that initiate a β turn or β pleated sheet, or, for example, are cyclized by the presence of disulfide bonded Cys residues) or unconstrained (that is, linear) amino acid sequences of about 15 amino acid residues, or less than about 15 amino acid residues.

[0102] Substitutes for an amino acid within the peptide sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline,

phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures include phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine and lysine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.

[0103] A conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity, or function of the resulting protein. For example, the peptide of the present disclosure comprises one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.

[0104] Particularly preferred amino acid substitutions include: a) Ala for Glu or vice versa, such that a negative charge may be reduced; b) Lys for Arg or vice versa, such that a positive charge may be maintained; c) Ala for Arg or vice versa, such that a positive charge may be reduced;

d) Glu for Asp or vice versa, such that a negative charge may be maintained;

e) Ser for Thr or vice versa, such that a free -OH can be maintained;

f) Gin for Asn or vice versa, such that a free NH2 can be maintained;

g) He for Leu or for Val or vice versa, as roughly equivalent hydrophobic amino acids; h) Phe for Tyr or vice versa, as roughly equivalent aromatic amino acids; and i) Ala for Cys or vice versa, such that disulphide bonding is affected.

[0105] Substitutes for an amino acid within the peptide sequence may be selected from any amino acids, including, but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine, N-formyl-L-methionine, sarcosine, or other N-methylated amino acids. In some embodiments, sarcosine substitutes for an amino acid within the peptide sequence.

[0106] In one embodiment, the invention discloses synthetic peptides derived from human astrovirus coat protein, the peptides comprising the amino acid sequences and modifications of SEQ ID NOs: 3-47. In some embodiments, the invention discloses synthetic peptides derived from human astrovirus coat protein, the peptides comprising the amino acid sequences and modifications of SEQ ID NOs: 3 and 19-29. In others embodiments, the invention discloses synthetic peptides derived from human astrovirus coat protein, the peptides comprising the amino acid sequences and modifications of SEQ ID NOs: 4-18 and 30-47.

[0107] In another embodiment, the invention discloses a synthetic peptide comprising the amino acid sequence of SEQ ID NO: 3, with one or more amino acid substitutions, modifications, insertions, or deletions, wherein the peptide regulates complement activation and/or has anti-microbial activity, amongst other therapeutic activities described herein.

[0108] In another embodiment, the invention discloses a synthetic peptide comprising the amino acid sequence of SEQ ID NO: 3, with one or more sarcosine substitutions, wherein the peptide regulates complement activation and/or has anti-microbial activity amongst other therapeutic activities described herein.

[0109] In another embodiment, the invention discloses a synthetic peptide comprising the amino acid sequence of SEQ ID NO: 3, with one or more alanine substitutions, wherein the peptide regulates complement activation and/or has anti-microbial activity amongst other therapeutic activities described herein.

[0110] In another embodiment, the invention discloses a synthetic peptide comprising the PEGylated amino acid sequence of SEQ ID NO: 3, wherein the peptide regulates complement activation and/or has anti-microbial activity amongst other therapeutic activities described herein.

[0111] The peptide compounds may have internal peptide deletions and substitutions as well as deletions and substitutions at the N-terminus and C-terminus based on SEQ ID NO: 3. In some embodiments, the peptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, modifications, insertions, or deletions.

[0112] In some embodiments, the peptide sequence has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%), at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 3.

[0113] In some embodiments, the present invention relates to therapeutically active peptides having the effects of treating or preventing organ dysfunction induced by ischemia, inflammation and/or toxic effects of poising or drug treatment. In other embodiments, the present invention also relates to therapeutically active peptides having anti-microbial effects. In other embodiments, the present invention relates to therapeutically active peptides having the effect of treating cystic fibrosis. In other embodiments, the present invention relates to therapeutically active peptides having the effect of treating or preventing acute intravascular hemolytic transfusion reactions (ATHTRs). In other embodiments, the present invention relates to therapeutically active peptides having the effect of treating birth asphyxia. The present invention also relates to therapeutically active peptides having the effect of treating acute complement-mediated diseases.

[0114] As used herein, a peptide sequence is "therapeutically active" if it can be used for the treatment, remission, or attenuation of a disease state, physiological condition, symptoms or etiological indication(s) or evaluation or diagnosis thereof. A peptide sequence is

"prophylactically active" if it can be used to prevent a disease state, physiological condition, symptoms or etiological indications.

[0115] The term "subject," as used herein, means any subject for whom diagnosis, prognosis, or therapy is desired. For example, a subject can be a mammal, e.g., a human or non-human primate (such as an ape, monkey, orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

[0116] As used herein, "treat," "treating," or "treatment" refers to administering a therapy in an amount, manner (e.g., schedule of administration), and/or mode (e.g., route of administration), effective to improve a disorder (e.g., a disorder described herein) or a symptom thereof, or to prevent or slow the progression of a disorder (e.g., a disorder described herein) or a symptom thereof. This can be evidenced by, e.g., an improvement in a parameter associated with a disorder or a symptom thereof, e.g., to a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject. By preventing or slowing progression of a disorder or a symptom thereof, a treatment can prevent or slow deterioration resulting from a disorder or a symptom thereof in an affected or diagnosed subject.

Astrovirus Coat Protein Peptides and Derivatives

[0117] CP1 is a peptide derived from human astrovirus coat protein, the peptide comprising an amino acid sequence of SEQ ID NO: 1.

[0118] Using CP1 as the parent peptide, internal deletions of residue 8 to residue 22 were made for the Δ8-22 peptide (SEQ ID NO: 2) (internal deletions are shown as dashes in TABLE 1). This peptide was active in all functional assays tested and bound Clq. The 15 amino acid residues from Δ8-22 peptide were scrambled to generate the Polar Assortant peptide (SEQ ID NO. 3). The scrambled Polar Assortant peptide (SEQ ID NO: 3) is also referred to as PA, PICl, AstroFend, or AF. As discussed herein, the term "PICl" peptide includes peptides with the amino acid sequence set forth in SEQ ID NO: 3 as well as peptides with the same amino acid sequence but with modifications such as PEGylation. The PA peptide was active in all functional assays tested. A series of other peptide deletions, substitutions, and modifications of CP1 are described in US Patent No. 8,906,845, the contents of which are hereby incorporated by reference in its entirety.

[0119] A series of peptide substitutions, and modifications of PA are disclosed, as shown in TABLES 1, 2, 3, and 4 below. This application discloses synthetic peptides comprising any one of the amino acid sequences of SEQ ID NOs: 1-47, as shown in TABLES 1-4. As therapeutic agents for humans, the route of administration for the peptide compounds may be, for example, topical, enteral, inhaled, parenteral (i.e. intramuscular, subcutaneous, intravenous), or administered through a nebulizer, as well as any other routes of

administration known in the art.

TABLE 1

Peptide Peptide sequence (N— » C)

CPl PAICQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO:i;

Δ8-22 PAICQRA EIEACILL (SEQ ID NO : 2 ]

Polar Assortant (also

known as PA, PIC 1, IALILEPICCQERAA (SEQ ID NO : 3 )

AstroFend, or AF)

Sarcosine Substitutions

[0120] Sarcosine ("Sar") is a natural amino acid, also known as N-methylglycine.

Sarcosine is an intermediate in the metabolism of choline to glycine. Sarcosine substitution was used to substitute each residue of PA sequentially (TABLE 2). Sarcosine substitution can be used to identify a peptide that retains complement inhibiting activity and is soluble in water.

[0121] Disclosed herein are peptide compounds substituted with sarcosine at certain positions. Table 2 discloses synthetic peptides comprising the sequence of SEQ ID NO: 3, wherein one or more of the amino acids are substituted with sarcosine, and wherein the peptides regulate complement activation and/or have anti-microbial activity. In one or more embodiments, the amino acids substituted with sarcosine are at positions 7, or 9. In one or more embodiments, two or more of the amino acids are substituted with sarcosine.

TABLE 2

Peptide Peptide Amino Acid

SEQ ID NO

Name Sequence

PA IALILEPICCQERAA SEQ ID NO: 3

PA-IlSar (Sar) ALILEPICCQERAA SEQ ID NO: 4

PA-A2Sar I (Sar) LILEPICCQERAA SEQ ID NO: 5

PA-L3Sar IA(Sar) ILEPICCQERAA SEQ ID NO: 6

PA-I4Sar IAL (Sar) LEPICCQERAA SEQ ID NO: 7

PA-L5Sar IALI (Sar) EPICCQERAA SEQ ID NO: 8 PA-E6Sar IALIL (Sar) PICCQERAA SEQ ID NO : 9

PA-P7Sar IALILE (Sar) ICCQERAA SEQ ID NO: 10

PA-I8Sar IALILEP (Sar) CCQERAA SEQ ID NO: 11

PA-C9Sar IALILEPI (Sar) CQERAA SEQ ID NO: 12

PA-C10Sar IALILEPIC (Sar) QERAA SEQ ID NO: 13

PA-Ql l Sar ALILEPICC (Sar) ERAA SEQ ID NO: 14

PA-E12Sar IALILEPICCQ (Sar) RAA SEQ ID NO: 15

PA-R13Sar IALILEPICCQE (Sar) AA SEQ ID NO: 16

PA-A14Sar IALILEPICCQER (Sar) A SEQ ID NO: 17

PA-A15Sar IALILEPICCQERA ( Sar ) SEQ ID NO: 18

PEGylation

[0122] Polyethylene glycol (PEG) is an oligomer or polymer of ethylene oxide.

Disclosed herein are peptide compounds that are PEGylated (i.e. have one or more PEG moieties attached). PEGylation can be used to identify a modified peptide that retains complement inhibiting activity and is soluble in water. One or more PEG moieties can be attached to the N terminus of the peptide, the C terminus of the peptide, or to both the N and C terminus of the peptide. The PEGylated peptide compound includes any of the peptides described herein (Tables 1-4) wherein the peptide is modified through PEGylation of the N terminus, the C terminus, or both the N terminus and C terminus.

[0123] PEGylation was used to attach one or more PEG moieties to the N terminus, the C terminus, or both the N terminus and C terminus of PA (TABLE 3). In one or more embodiments, 24 PEG moieties are attached to the N terminus of PA. In one or more embodiments, 24 PEG moieties are attached to the C terminus of PA. In one or more embodiments, 24 PEG moieties are attached to the N terminus of PA and to the C terminus of PA. In one or more embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more PEG moieties are attached to the N terminus of PA. In one or more embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 PEG moieties were attached to the C terminus of PA. In one or more embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more PEG moieties are attached to both the N terminus and the C terminus of PA. [0124] Disclosed herein are PEGylated peptide compounds. This application discloses synthetic peptides comprising the sequence of SEQ ID NO: 3 (PICl), wherein one or more PEG moieties are attached, and wherein the peptides regulate complement activation and/or have anti-microbial activity.

TABLE 3

Peptide Name Peptide Amino Acid Sequence SEQ ID NO dPEG24-PA-dPEG24 dPEG24-IALILEPICCQERAA-dPEG24 SEQ ID NO : : 1 9 dPEG24-PA dPEG24- IALILEPICCQERAA SEQ ID NO : : 2 0

PA-dPEG24 IALILEPICCQERAA-dPEG24 SEQ ID NO : : 2 1

PA-dPEG20 IALILEPICCQERAA-dPEG20 SEQ ID NO : : 22

PA-dPEG16 IALILEPICCQERAA-dPEGl 6 SEQ ID NO : : 23

PA-dPEG12 IALILEPICCQERAA-dPEG12 SEQ ID NO : : 24

PA-dPEG08 IALILEPICCQERAA-dPEGO 8 SEQ ID NO : : 25

PA-dPEG06 IALILEPICCQERAA-dPEGO 6 SEQ ID NO : : 2 6

PA-dPEG04 IALILEPICCQERAA-dPEGO4 SEQ ID NO : : 2 7

PA-dPEG03 IALILEPICCQERAA-dPEGO3 SEQ ID NO : : 2 8

PA-dPEG02 IALILEPICCQERAA-dPEGO2 SEQ ID NO : : 2 9

PEGylation of Peptides in Combination with Amino Acid Substitutions

[0125] This application discloses PA/PIC 1 that has been modified by a combination of sarcosine substitutions and/or alanine substitutions and/or PEGylation. A series of peptide substitutions and modifications of PA are disclosed, as shown in TABLE 4 below. The modified and substituted peptides can be used to identify a peptide that retains complement inhibiting activity and is soluble in water. This application discloses a synthetic peptide comprising the sequence of SEQ ID NO: 3, wherein one or more of the amino acids are substituted with sarcosine or alanine, wherein the peptides regulate complement activation and/or has anti-microbial activity. This application also discloses a synthetic peptide comprising the sequence of SEQ ID NO: 3, wherein one or more of the amino acids are substituted with sarcosine or alanine and one or more PEG moieties is attached to the N terminus of the peptides, the C terminus of the peptides, or to both the N and C terminus of the peptides, wherein the peptides regulate complement activation and/or have anti-microbial activity.

[0126] In one or more embodiments, two or more of the amino acids are substituted with sarcosine. In one or more embodiments, two or more of the amino acids are substituted with alanine. In one or more embodiments, one or more of the amino acids are substituted with sarcosine and one or more of the amino acids are substituted with alanine. The substituted peptides can further be modified by PEGylation as described above.

TABLE 4

Peptide Name and

Peptide Amino Acid Sequence SEQ ID NO

Controls

Water

DMSO

PA-dPEG24 IALILEPICCQERAA-dPEG24 SEQ ID NO: :21

PA-C9SarC10A IALILEPI (Sar)AQERAA SEQ ID NO: : 30

PA-C9SarD10 IALILEPI (Sar) QERAA SEQ ID NO: : 31

PA-P7SarC9Sar IALILE (Sar) I (Sar) CQERAA SEQ ID NO: : 32

PA-E6Sar-dPEG24 IALIL (Sar) PICCQERAA-

SEQ ID NO: : 33 dPEG24

PA-Ql l Sar-dPEG24 IALILEPICC (Sar) ERAA-

SEQ ID NO: : 34 dPEG24

PA-R13Sar-dPEG24 IALILEPICCQE (Sar) AA-

SEQ ID NO: : 35 dPEG24

PA-A14Sar-dPEG24 IALILEPICCQER (Sar) A-

SEQ ID NO: : 36 dPEG24

E6SarP7Sar IALIL (Sar) (Sar) ICCQERAA SEQ ID NO: : 37

E6SarC9Sar IALIL (Sar) PI (Sar) CQERAA SEQ ID NO: : 38

Ql l SarP7Sar IALILE (Sar) ICC (Sar) ERAA SEQ ID NO: : 39

Ql l SarC9Sar IALILEPI (Sar) C (Sar) ERAA SEQ ID NO: : 40

R13SarP7Sar IALILE (Sar) ICCQE (Sar) AA SEQ ID NO: : 41

R13SarC9Sar IALILEPI (Sar) CQE (Sar) AA SEQ ID NO: : 42

A14SarP7Sar IALILE (Sar) ICCQER (Sar) A SEQ ID NO: : 43

A14SarC9Sar IALILEPI (Sar) CQER (Sar) A SEQ ID NO: : 44

E6AE12A-dPEG24 IALILAPICCQARAA-dPEG24 SEQ ID NO: : 45

E6AE12AC9Sar IALILAPI (Sar) CQARAA SEQ ID NO: : 46

E6AE12AP7Sar IALILA (Sar ) ICCQARAA SEQ ID NO: : 47 Antimicrobial Activity of PIC 1 Peptides

[0127] There is currently a critical need for new antibiotics as many microorganisms have become resistant to currently prescribed antibiotics. Additionally, most antibiotics are derived from other microbial organisms that bacteria have competed against for space and energy over the millennia, leading to rapid and predictable emergence of resistance.

[0128] The CP1 peptide was initially identified by its weak homology to the human neutrophil defensing peptide 1 (HNP-1). In addition to inhibiting complement activation, HNP-1 has the ability to inhibit bacterial growth. The PIC 1 peptides described herein, including SEQ ID NOs: 3-47, are very different in amino acid sequence to HNP-1 and have no known homologs in nature. Surprisingly, in some aspects, the PICl peptides have antibacterial activity.

[0129] In some aspects, the disclosed peptide compounds have a direct antimicrobial effect and are, thus, ideal for inhibiting the growth of bacterial diseases. The disclosed peptide compounds can be used to prevent and treat diseases mediated by bacteria. In some embodiments, the disclosed peptide compounds can be used to prevent and treat Gram positive and Gram negative bacterial infections. In some embodiments, the disclosed peptide compounds can be used to prevent and treat, for example, Pseudomonas aeruginosa, MRSA, and carbapenemase-resistant enterobacteriacea (CREs) (e.g. resistant Klebsiella pneumonia). The disclosed peptide compounds can be used to prevent and treat bacterial vaginosis and vaginitis. In one or more embodiments, the disclosed peptide compounds kill the causative organism of bacterial vaginosis and also blocks the inflammation, which disrupts barrier defenses increasing risk of HIV transmission. As the PICl peptides have no homology with known proteins or peptides, this has the potential to decrease the likelihood of emergence of resistance.

[0130] The disclosed peptide compounds can also enhance growth of Lactobacillus. In some embodiments of the invention, L. acidophilus and L. leichmannii are enhanced by the disclosed peptide compounds. In certain aspects, the invention provides a method of enhancing Lactobacillus growth by administering a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOS: 3-47.

[0131] Bacterial vaginosis and vaginitis are currently treated with systemic antibiotics. The disclosed peptide compounds can be used to prevent and treat bacterial vaginosis and vaginitis by local administration (e.g., topical administration) or by systemic administration (e.g., intravenous administration).

[0132] In certain aspects, the invention provides a method of treating a bacterial infection comprising administering to a subject a pharmaceutical composition comprising a

therapeutically effective amount of a synthetic peptide comprising the amino acid sequence and modifications selected from SEQ ID NOs: 3-47. In one or more embodiments, the bacteria are Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, or Gardnerella sp. In one or more embodiments, the subject has cystic fibrosis, and the peptide compounds treat cystic fibrosis. In one or more embodiments, the subject has gonorrhea or chlamydia, and the peptide compounds treat gonorrhea or chlamydia. In one or more embodiments, the subject has pneumonia, and the peptide compounds treat pneumonia.

[0133] In certain aspects, the invention provides a method of treating bacterial vaginitis comprising administering to a subject a pharmaceutical composition comprising a

therapeutically effective amount of a synthetic peptide comprising the amino acid sequence and modifications selected from SEQ ID NOs: 3-47.

The Complement System and Diseases Associated with Its Dysregulation

[0134] While complement is a vital host defense against microorganisms such as bacteria and some enveloped viruses, its unchecked activation can cause devastating host cell damage. Host tissue damage mediated by complement has been implicated in a wide variety of diseases, including autoimmune pathologies such as: rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, autoimmune hemolytic anemia, membranoproliferative glomerulonephritis, and serum sickness. It has also been identified as contributing to the development of the following diseases: Adult Respiratory Distress Syndrome (ARDS), ischemia-reperfusion injuries (including stroke and myocardial infarction), alio- and xeno- transplantation complications (including hyperacute rejection and graft versus host disease (GVHD), Alzheimer's disease, burn injuries, hemodialysis damage, cardiopulmonary bypass damage, and paroxysmal nocturnal hemoglobinuria (PNH).

[0135] Hereditary angioedema (HAE) is a rare genetic disorder caused by reduced levels of or non-functional CI -inhibitor; symptoms of HAE include acute edema. CI -inhibitor naturally regulates CI activation, and treatment of acute edema requires substantial infusion of CI -inhibitor or plasma transfusion. Because astrovirus CP functionally blocks CI activation, using the disclosed peptide compounds to treat HAE fulfills a therapeutic need because CI -inhibitor has to be purified from human sera from multiple subjects and, therefore, could be contaminated with human blood-borne pathogens. Therapeutic administration of the disclosed peptide compounds regulates CI either in adjunct therapy with CI -inhibitor or as a stand-alone therapeutic treatment.

[0136] The disclosed peptide compounds can selectively regulate Clq and MBL activation without affecting alternative pathway activity and are, thus, ideal for preventing and treating diseases mediated by the dysregulated activation of the classical and lectin pathways. Specific blockade of classical and lectin pathways are particularly needed, as both of these pathways have been implicated in ischemia-reperfusion induced injury in many animal models. [Castellano et al., "Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage '' Am J Pathol. 2010; 176(4): 1648-59; Lee et al, "Early complement factors in the local tissue immunocomplex generated during intestinal ischemia/reperfusion injury." Mol. Immunol. 2010

Feb;47(5):972-81; Tjernberg, et al, "Acute antibody-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet

transplantation " Transplantation. 2008; Apr 27;85(8): 1193-9; Zhang et al. "The role of natural IgMin myocardial ischemia-reperfusion injury." J Mol Cell Cardiol. 2006

Jul;41(l):62-7). The alternative pathway is essential for immune surveillance against invading pathogens, and humans with alternative pathway defects suffer severe bacterial infections. By binding and inactivating Clq and MBL, the peptide compounds can efficiently regulate classical and lectin pathway activation while leaving the alternative pathway intact.

[0137] The term "regulate," as used herein, refers to i) controlling, reducing, inhibiting or regulating the biological function of an enzyme, protein, peptide, factor, byproduct, or derivative thereof, either individually or in complexes; ii) reducing the quantity of a biological protein, peptide, or derivative thereof, either in vivo or in vitro; or iii) interrupting a biological chain of events, cascade, or pathway known to comprise a related series of biological or chemical reactions. The term "regulate" may thus be used, for example, to describe reducing the quantity of a single component of the complement cascade compared to a control sample, reducing the rate or total amount of formation of a component or complex of components, or reducing the overall activity of a complex process or series of biological reactions, leading to such outcomes as cell lysis, formation of convertase enzymes, formation of complement-derived membrane attack complexes, inflammation, or inflammatory disease. In an in vitro assay, the term "regulate" may refer to the measurable change or reduction of some biological or chemical event, but the person of ordinary skill in the art will appreciate that the measurable change or reduction need not be total to be "regulatory."

[0138] In one or more aspects, the disclosed peptide compounds can be used to treat an inflammatory disease. In one or more embodiments, the disclosed peptide compounds can be used to treat an autoimmune disease. In one or more embodiments, the disclosed peptide compounds can be used to treat hemolytic transfusion reactions, cold-agglutinin disease, immune-complex diseases (e.g. serum sickness), thalassemia, sickle cell disease, ABO incompatibility (e.g. neonatal jaundice), acute/hyperacute solid organ transplantation rejection, solid organ transplantation warm/cold ischemia, instant blood-mediated

inflammatory reaction (IBMIR), systemic lupus erythematosus (SLE), rheumatoid arthritis, ischemia-reperfusion injury, myocardial infarct, stroke, hypoxic ischemic encephalopathy (e.g. birth asphyxia brain injury), traumatic brain injury, coronary artery bypass surgery, cystic fibrosis, wound healing, cancer, Alzheimer's disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (P H), atypical hemolytic uremic syndrome (aHUS), asthma, chronic obstructive pulmonary disease (COPD), Crohn's disease, Sepsis

syndrome/ ARDS/SIRS, glomerulonephritis (e.g. lupus nephritis, anti-glomerular basement membrane disease, anti-neutrophil cytoplasmic autoantibody-induced, membranoproliferative glomerulonephritis (e.g. dense deposit disease), membranous nephropathy), IgA nephropathy, or C3 glomerulopathy.

Complement-Mediated Acute Intravascular Hemolytic Transfusion Reaction (AIHTR)

[0139] Acute intravascular hemolytic transfusion reaction ("ATR," "AHTR," or

"AIHTR") is a type of transfusion reaction that is associated with hemolysis. AIHTR can result in complement mediated lysis and rapid destruction of donor red blood cells. AIHTRs have a broad clinical presentation from mild and transitory signs and symptoms to serious cases of shock, disseminated intravascular coagulation, renal failure and death.

[0140] The American Red Cross estimates that 30 million blood components are transfused every year in the United States (US) [Barbee I. Whitaker PSH, PhD, The 2011 National Blood Collection and Utilization Survey Report; 2011]. Approximately 70,000 US patients living with sickle cell disease and 1.6 million individuals diagnosed with cancer require blood transfusions routinely as part of their disease management. Blood transfusions are life-saving, but carry the risk of a variety of reactions some of which are potentially life- threatening such as acute intravascular hemolytic transfusion reactions (AIHTRs) (Murphy MF, Waters JH, Wood EM, Yazer MH. "Transfusing blood safely and appropriately." BMJ. 2013;347:f4303]. AIHTR is estimated to occur in one-fifth of total transfusions (Refaai MA, Blumberg N. "The transfusion dilemma—weighing the known and newly proposed risks of blood transfusions against the uncertain benefits." Best practice & research Clinical anaesthesiology. 2013;27(1): 17-35). Individuals receiving frequent blood transfusions will develop alloantibodies and autoantibodies to red blood cell (RBC) antigens over time making cross-matching increasingly difficult and thus increasing the risk of AIHTR (Aygun B, Padmanabhan S, Paley C, Chandrasekaran V. "Clinical significance of RBC alloantibodies and autoantibodies in sickle cell patients who received transfusions." Transfusion.

2002;42(l):37-43]. Current transfusion safe-guards include 'typing', 'antibody screening' as well as 'cross matching', and while these measures have made transfusions safer than ever before, transfusion reactions still occur (Osterman JL, Arora S. "Blood product transfusions and reactions." Emergency medicine clinics of North America. 2014;32(3):727-738]. AIHTR occurs when host antibodies bind to the transfused erythrocytes initiating classical complement pathway activation leading to the generation of the inflammatory mediators C3a and C5a as well as C3b opsonization and hemolysis of the transfused cells via the membrane attack complex (MAC) (Stowell SR, Winkler AM, Maier CL, et al. "Initiation and regulation of complement during hemolytic transfusion reactions." Clinical & developmental immunology. 2012;2012:307093]. While the role of complement in AIHTR is well recognized, to date, only one case has been reported describing clinical intervention of an AIHTR by inhibiting generation of the complement anaphylatoxins C3a and C5a [Weinstock C, Mohle R, Dorn C, et al. "Successful use of eculizumab for treatment of an acute hemolytic reaction after ABO-incompatible red blood cell transfusion." Transfusion. 2015;55(3):605- 610].

[0141] The classical pathway of complement acts as an amplification cascade after initial activation of complement complex CI by antibodies (Frank MM AJ ed Complement system. In: Austen KF, Atkinson JP, Cantor HI ed. Samter's Immunologic Disease. New York:

Lippincott Williams and Wilkins; 2001]. The CI complex consists of the pattern recognition molecule Clq and the serine protease tetramer Clr-Cls-Cls-Clr. Upon binding of Clq to IgM or clustered IgG antibodies, Clq undergoes a conformational change to activate Clr- Cls-Cls-Clr and initiate classical pathway-mediated complement activation. The Peptide Inhibitor of Complement CI (PICl) described herein is a peptide inhibitor of the classical pathway of complement (Mauri ello CT, Pallera HK, Sharp JA, et al. "A novel peptide inhibitor of classical and lectin complement activation including ABO incompatibility." Mol Immunol. 2013;53(l-2): 132-139; Sharp J A, Whitley PH, Cunnion KM, Krishna NK. "Peptide inhibitor of complement CI, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential." Frontlmmunol . 2014;5:406; Gronemus JQ, Hair PS, Crawford KB, Nyalwidhe JO, Cunnion KM, Krishna NK. "Potent inhibition of the classical pathway of complement by a novel Clq-binding peptide derived from the human astrovirus coat protein." Mol Immunol. 2010;48(l-3):305-313). PICl inhibits antibody-initiated activation of CI by binding Clq and preventing activation of Clr-Cls-Cls-Clrl2. PICl also has also been shown to inhibit classical complement pathway-mediated ABO incompatible hemolysis of human erythrocytes in vitro (Mauriello CT, Pallera HK, Sharp JA, et al. "A novel peptide inhibitor of classical and lectin complement activation including ABO incompatibility." Mol Immunol. 2013;53(l-2): 132-139; Sharp JA, Whitley PH, Cunnion KM, Krishna NK. "Peptide inhibitor of complement CI, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential." Frontlmmunol20\4,5 :406; Shah TA, Mauriello CT, Hair PS, et al. "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis." Transfusion. 2014). When the aqueous polyethylene glycol (PEG)-conjugated version of the PICl, PA-dPEG24 (SEQ ID NO: 21), described herein is administered intravascularly into rats, it can achieve greater than 90% systemic inhibition of the animal's serum complement levels within 30 seconds (Sharp JA, Whitley PH, Cunnion KM, Krishna NK. "Peptide inhibitor of complement CI, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential." Front Immunol . 2014;5:406]. The ability of PICl to inhibit complement activation makes it an ideal molecule to block the rapid complement-mediated hemolysis that typifies AIHTR.

[0142] A rat AIHTR disease model based on xenotransfusion of human RBCs was established (Shah TA, Mauriello CT, Hair PS, et al. "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis." Transfusion. 2014]. Wistar rats have natural hemagglutinins in their serum that will bind to human AB erythrocytes (Shah et al; Aptekman PM, Bogden AE. "Characterization of the natural hemagglutinins in normal rat serum associated with a negative phase following tumor implantation." Cancer Research. 1956; 16(3):216-221). Upon transfusion of human erythrocytes, they will produce a rapid ABO incompatibility-like hemolysis via antibody- initiated classical complement pathway activation (Yazdanbakhsh K, Kang S, Tamasauskas D, Sung D, Scaradavou A. "Complement receptor 1 inhibitors for prevention of immune- mediated red cell destruction: potential use in transfusion therapy." Blood.

2003; 101(12):5046-5052].

[0143] PICl peptides, including PA (SEQ ID NO: 3) and PA-dPEG24 (SEQ ID NO: 21), can block complement-mediated lysis of AB human red blood cells (RBC) by O serum in vitro. This assay mimics ABO incompatibility. PA-dPEG24 has efficacy in a rodent model of AIHTR demonstrating inhibition of human RBC lysis in a prevention and rescue scenario. Thus PICl peptides can be used in the treatment of transfusion reactions in humans for which no current therapy exists.

[0144] Current blood banking organization or transfusion medicine practice does not have a method to directly evaluate risk for complement mediated RBC lysis between donor and recipient. The disclosed peptide compounds can be used as a diagnostic tool. The disclosed peptide compounds can also be used as a prophylactic treatment to prevent AIHTR or as a rescue treatment during an AIHTR has significant clinical implications. The disclosed peptide compounds can prevent the high concentrations of free hemoglobin that cause acute kidney injury in AIHTR. There are currently no therapies for ATRs other than supportive care.

[0145] The disclosed peptide compounds can be used to detect complement-mediated lysis of RBCs and are thus ideal for discriminating if an erythrocyte transfusion may cause AIHTR. The disclosed peptide compounds can be used to predict AIHTR.

[0146] In certain aspects, the invention provides a method of treating acute transfusion reaction comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a synthetic peptide comprising the amino acid sequence and modifications selected from SEQ ID NOs: 3-47. In one or more embodiments, the pharmaceutical composition is administered before the subject is administered a blood transfusion. In one or more embodiments, the pharmaceutical composition is administered after the subject is administered a blood transfusion. In one or more embodiments, the pharmaceutical composition is administered before and after the subject is administered a blood transfusion.

Complement Effectors in Cystic Fibrosis (CF) [0147] In cystic fibrosis (CF), lung damage is believed to be mediated by a cycle of obstruction, infection, and inflammation. It has been determined that complement effectors are elevated in the lung fluids of CF patients compared to normal controls.

[0148] Cystic fibrosis (CF) afflicts 30,000 individuals in the United States (Boyle MP. "Adult cystic fibrosis." JAMA 2007; 298(15): 1787-93) with respiratory failure causing the majority of deaths. Progressive destruction of lung parenchyma is mediated by a cycle of obstruction, infection with bacterial pathogens, and inflammation (Rowe SM, Miller S, Sorscher EJ, "Cystic fibrosis." N Engl J Med 2005; 352(19): 1992-2001). As the cycle repeats there is progression from lung damage to lung scarring and finally pulmonary failure (Gibson RL, Burns JL, Ramsey BW. "Pathophysiology and management of pulmonary infections in cystic fibrosis." Am J Respir Crit Care e<f 2003; 168(8):918-51].

[0149] The most destructive inflammatory cascade in the human body is the complement system, which contributes to host tissue damage in numerous inflammatory disease processes (Ricklin D, Lambris JD. "Complement-targeted therapeutics." Nat Biotechnol 2007;

25(11): 1265-75). Recent evidence has shown that complement proteins are major constituents of lung fluid in CF patients and normal humans, where C3 and C4 account for two of the four most prevalent proteins (Gharib SA, Vaisar T, Aitken ML, et al. "Mapping the lung proteome in cystic fibrosis." J Proteome Res 2009;8(6):3020-8). This suggests that complement may play a much larger role in CF lung inflammation than what has been previously suspected. Antibody binding to bacteria can activate the classical complement pathway via the initiating component CI (FIG. 9). C4 is a cascade component for the classical (i.e. antibody initiated) pathway leading to formation of the opsonin C4b and downstream activation of C3 (Lambris JD, Sahu A, Wetsel RA. "The chemistry and biology of C3, C4, and C5." In: Volanakis JE, Frank MM, (eds). The human complement system in health and disease. New York: Marcel Dekker; 1998, 83 -118.). C3 is the central

complement component, which upon activation generates the complement effector C3a and covalently binds cells with the opsonic fragments C3b and iC3b. C3b then initiates activation of C5, generating the extremely potent anaphylatoxin C5a. C5a is among the most powerful stimulants for neutrophil migration and activation, leading to oxidative burst and

degranulation (Lambris JD, Sahu A, Wetsel RA. "The chemistry and biology of C3, C4, and C5." In: Volanakis JE, Frank MM, (eds). The human complement system in health and disease. New York: Marcel Dekker; 1998, 83 -118; Tralau T, Meyer-Hoffert U, Schroder JM, et al. "Human leukocyte elastase and cathepsin G are specific inhibitors of C5a-dependent neutrophil enzyme release and chemotaxis." Exp Dermatol 2004; 13(5):316-25). Neutrophil death, which follows degranulation, is a major source of the viscous DNA that contributes to the obstruction of airways in the CF lung (Dwyer M, Shan Q, D'Ortona S, et al. "Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor." J Innate Immun 2014; Hodson ME. "Aerosolized dornase alfa (rhDNase) for therapy of cystic fibrosis." Am J Respir Crit Care Med 1995; 151(3 Pt 2):S70-4]. Among the neutrophil granule products released is neutrophil elastase, which is a major contributor to parenchymal lung damage in CF (Gifford AM, Chalmers JD. "The role of neutrophils in cystic fibrosis." Curr Opin Hematol 2014;21(1): 16-22; Le Gars M, Descamps D, Roussel D, et al. "Neutrophil elastase degrades cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo '' Am J Respir Crit Care Med 2013; 187(2): 170-9; Sagel SD, Wagner BD, Anthony MM, et al. "Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis." Am J Respir Crit Care Med

2012; 186(9):857-65]. Thus, complement activation may play a significant role in neutrophil recruitment and activation in CF lungs, contributing to tissue damage. Additional properties of C5a that may also contribute to CF lung disease are stimulation of histamine release, enhancement of vascular permeability, and inducement of smooth muscle contraction (Lambris JD, Sahu A, Wetsel RA. "The chemistry and biology of C3, C4, and C5." In: Volanakis JE, Frank MM, (eds). The human complement system in health and disease. New York: Marcel Dekker; 1998, 83 -11]. The known inflammatory properties of C5a are consistent with the increasing evidence of the role of C5a in inflammatory lung diseases (Schmudde I, Strover HA, Vollbrandt T, et al. "C5a receptor signalling in dendritic cells controls the development of maladaptive Th2 and Thl7 immunity in experimental allergic asthma." Mucosal Immunol 2013; 6(4):807-25; Bosmann M, Ward PA. "Role of C3, C5 and anaphylatoxin receptors in acute lung injury and in sepsis." Adv Exp Med Biol 2012;

946: 147-59], including acute lung injury. Thus, although not bound by any theory, multiple lines of reasoning suggest that complement-mediated inflammation may be a major contributor to inflammatory lung damage in CF.

[0150] Some investigation into the potentially important role of C5a in the CF lung has been performed. In 1986, Fick et al. described the presence of increased amounts of C5a, measured by radioimmunoassay in the bronchoalveolar lavage (BAL) of nine CF patients with clinically stable lung disease compared with BAL from healthy controls. (Fick RB, Jr., Robbins RA, Squier SU, et al. "Complement activation in cystic fibrosis respiratory fluids: in vivo and in vitro generation of C5a and chemotactic activity." Pediatr Res 1986;

20(12): 1258-68). The CF BAL fluids were chemotactic for neutrophils, which appeared to correlate with C5a concentrations. The BAL fluids showed evidence of prior complement activation by the presence of C3c, assayed by crossed-immunoelectrophoresis. Two CF patients with the lowest C5a measurements were noted to have normal FEVi and FVC measurements, suggesting a potential association with lung damage. However, no further studies were then performed to test whether C5a concentrations in CF lung fluid correlate with either acute lung exacerbations or chronic lung disease progression in CF.

[0151] Although not bound by any mechanism, the disclosed peptide compounds can selectively regulate Clq and MBL activation without affecting alternative pathway activity and are, thus, ideal for treating cystic fibrosis. In one or more embodiments, the disclosed peptide compounds can be used to treat cystic fibrosis. In one or more embodiments, the disclosed peptide compounds can be used as anti-inflammatory and anti-microbial agents. In one or more embodiments, the disclosed peptide compounds can be used to treat cystic fibrosis by administrating the peptide(s) via nebulization directly into the lung. In one or more embodiments, administration of the disclosed peptide compound(s) via nebulization mitigates lung destruction caused by cystic fibrosis. The disclosed peptide compounds have clinical benefit for CF patients by slowing the progression of lung damage leading to increased life span and quality of life.

[0152] In certain aspects, the invention provides a method of treating cystic fibrosis comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence and modifications selected from SEQ ID NOs: 3-47.

Modulation of Clq interaction with Clq receptors

[0153] Clq interactions with Clq receptors appear to play important roles in homeostatic functions such as scavenging of apoptotic cellular debris and immune complexes as well as T cell signaling through antigen presenting cells (macrophage and dendritic cells). Currently, no clinical pharmacological agents modulate the interaction of Clq with Clq receptors.

[0154] The disclosed peptide compounds can be used to block Clq binding to Clq receptors, including calreticulin/cClqR. The ability of the disclosed peptide compounds to block binding of Clq to cellular receptors may have an important role in modulating intracellular signaling processes mediated by Clq binding to Clq receptors. The disclosed peptide compounds can be used to regulate the complement response in disease processes such as systemic lupus erythematosus and cancer.

Brain Damage in Birth Asphyxia

[0155] Complement activation is instrumental in the development of ischemia- reperfusion injuries such as neonatal hypoxic ischemic encephalopathy (HIE). Therapeutic hypothermia (HT) offers only an 11% reduction in death or disability. Published in vitro data has shown that HT paradoxically increase pro-inflammatory complement activation potentially limiting its benefit.

[0156] PICl peptides can reduce brain infarct volumes without prolonged systemic complement depletion. PICl peptides can be a useful adjunct to HT to improve neurological outcomes in HIE.

[0157] The disclosed peptide compounds can be us to prevent brain damage from birth asphyxia. The disclosed peptide compounds can also be used to prevent ischemia- reperfusion injury (IRI) in diseases such as myocardial infarct, coronary artery bypass surgery, stroke, etc. The disclosed peptide compounds can also be used to prevent hyperacute and acute solid organ transplantation rejection, which are classical complement pathway mediated events.

[0158] In certain aspects, the invention provides a method of treating birth asphyxia comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence and modifications selected from SEQ ID NOs: 3-47. In some embodiments, the subject is further treated by therapeutic hypothermia.

[0159] In certain aspects, the invention provides a method of treating hypoxic ischemic encephalopathy comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence and modifications selected from SEQ ID NOs: 3-47. In some embodiments, the subject is further treated by therapeutic hypothermia.

Myeloperoxidase (MPO) Activity

[0160] Myeloperoxidase (MPO) is an enzyme from neutrophils that creates hypochlorite (bleach) in acute inflammation and damages invading and host cells alike. This enzyme is known to be destructive to host tissues in Cystic Fibrosis (CF) and Hypoxic Ischemic Encephalopathy (HIE).

[0161] In some embodiments, PICl blocked the enzymatic activity of MPO in the sputum of cystic fibrosis patients. In some embodiments, PICl blocked enzymatic activity in brain tissue from HIE patents. In some embodiments, MPO activity present in the lysates of purified human neutrophils can be directly inhibited by PICl . In some embodiments, the invention demonstrates that PICl has anti -inflammatory activity against cystic fibrosis and HIE.

Autoimmune Hemolytic Anemia

[0162] The present disclosure provides peptide compounds capable of treating autoimmune hemolytic anemia. Autoimmune hemolytic anemia is a group of disorders characterized by a malfunction of the immune system that produces autoantibodies, which attack red blood cells as if they were substances foreign to the body. Autoimmune hemolytic anemia may be characterized by one or more of elevated serum bilirubin, excess urinary urobilinogen, reduced plasma haptoglobin, raised serum lactic dehydrogenase (LDH), hemosiderinuria, methemalbuminemia, spherocytosis, reticulocytosis, and/or erythroid hyperplasia of the bone marrow. In certain aspects of the invention, treating autoimmune hemolytic anemia comprises administering a peptide compounds selected from the group consisting of SEQ ID NO: 3-47.

Pharmaceutical Formulation and Administration

[0163] The present disclosure provides pharmaceutical compositions capable of regulating the complement system, comprising at least one peptide compound, as discussed above, and at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient. Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. They can be solid, semi-solid, or liquid. The pharmaceutical compositions of the present invention can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, or syrups.

[0164] Some examples of pharmaceutically acceptable carriers, diluents, stabilizers, or excipients include: lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The

pharmaceutical compositions of the present invention can be formulated using procedures known in the art to provide quick, normal, or sustained or delayed release of the active ingredient.

[0165] The disclosure relates to a method of regulating the complement system in a subject comprising administering to a subject the compositions described above. The pharmaceutical compositions of the present invention are prepared by mixing the peptide compound having the appropriate degree of purity with pharmaceutically acceptable carriers, diluents, or excipients. Examples of formulations and methods for preparing such

formulations are well known in the art. The pharmaceutical compositions of the present invention are useful as a prophylactic and therapeutic agent for various disorders and diseases, as set forth above. In one embodiment, the composition comprises a therapeutically effective amount of the peptide compound. In another embodiment, the composition comprises at least one other active ingredient effective in treating at least one disease associated with complement-mediated tissue damage. The term "therapeutically effective amount," as used herein, refers to the total amount of each active component that is sufficient to show a benefit to the subject.

[0166] The therapeutically effective amount of the peptide compound varies depending on several factors, such as the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound employed, the duration of treatment, the co-therapy involved, and the age, gender, weight, and condition of the subject, etc. One of ordinary skill in the art can determine the therapeutically effective amount. Accordingly, one of ordinary skill in the art may need to titer the dosage and modify the route of administration to obtain the maximal therapeutic effect.

[0167] The effective daily dose generally is within the range of from about 0.001 to about 200 milligrams per kilogram (mg/kg) of body weight, preferably about 80 to about 160 mg/kg, more preferably about 0.1 to about 20 mg/kg. This dose can be achieved through a 1- 6 time(s) daily dosing regimen. Alternatively, optimal treatment can be achieved through a sustained release formulation with a less frequent dosing regimen.

[0168] Pharmaceutical formulations may be adapted for administration by any

appropriate route, for example, by the oral, nasal, topical (including buccal, sublingual, or transdermal), or parenteral (including subcutaneous, intracutaneous, intramuscular, intraarticular, intraperitoneal, intrasynovial, intrasternal, intrathecal, intralesional,

intravenous, or intradermal injections or infusions) route. For human administration, the formulations preferably meet sterility, pyrogenicity, general safety, and purity standards, as required by the offices of the Food and Drug Administration (FDA).

Combination Therapies

[0169] A further embodiment of the invention provides a method of preventing or treating a disease associated with complement-mediated tissue damage, comprising administering to a subject a pharmaceutical composition of the present invention. While the pharmaceutical compositions of the present invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more therapeutic or prophylactic agent(s) that is(are) effective for preventing or treating the disease. In this aspect, the method of the present invention comprises administrating a pharmaceutical composition of the present invention before, concurrently, and/or after one or more additional therapeutic or prophylactic agents effective in treating at least one disease associated with complement-mediated tissue damage.

[0170] For example, the pharmaceutical compositions of the present invention can be used to treat brain asphyxia or hypoxic ischemic encephalopathy, either alone or in combination with therapeutic hypothermia.

[0171] For example, the pharmaceutical compositions of the present invention can be used to treat rheumatoid arthritis, either alone or in combination with a non-steroidal antiinflammatory agent (NSAID), a corticosteroid, or a disease modifying anti -rheumatic drug (DMARD).

[0172] Examples of NSAIDs include: salicylates (such as aspirin, amoxiprin, benorilate, choline magnesium salicylate, diflunisal, faislamine, methyl salicylate, magnesium salicylate, and salicyl salicylate (salsalate)), arylalkanoic acids (such as diclofenac, aceclofenac, acemetacin, bromfenac, etodolac, indometacin, ketorolac, nabumetone, sulindac, and tolmeti), 2-arylpropionic acids (such as ibuprofen, carprofen, fenbufen, fenoprofen, flurbiprofen, ketoprofen, loxoprofen, naproxen, tiaprofenic acid, and suprofen), N- arylanthranilic acids (such as mefenamic acid and meclofenamic acid), pyrazolidine derivatives (such as phenylbutazone, azapropazone, metamizole, oxyphenbutazone, and sulfinprazone), oxicams (such as piroxicam, lornoxicam, meloxicam, and tenoxicam), COX-2 inhibitors (such as etoricoxib, lumiracoxib, and parecoxib), sulphonanilides such as nimesulide, and others such as licofelone and omega-3 fatty acids. [0173] Examples of corticosteroids include: triamcinolone (Aristocort®), cortisone (Cortone® Acetate Tablets), dexamethasone (Decadron® Elixir), prednisone (Deltasone®), and methylprednisolone (Medrol®).

[0174] Examples of DMARDs include: methotrexate (Rheumatrex®), leflunomide (Arava®), etanercept (Enbrel®), infliximab (Remicade®), adalimumab (Humira®), anakinra (Kineret®), sulfasalazine (Azulfidine EN-Tabs®), antimalarials, gold salts, d-penicillamine, cyclosporin A, cyclophosphamide and azathioprine.

[0175] Soliris™ (eculizumab) is a humanized anti-C5 monoclonal antibody. It has been approved by the FDA for the treatment of the rare form of hemolytic anemia, paroxysmal nocturnal hemoglobinuria. In one embodiment, the pharmaceutical compositions of the present invention can be used in combination with Soliris™ in treating paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, heart disease, pulmonary diseases, autoimmune diseases, asthma, as well as the ancillary care of transplants.

[0176] The pharmaceutical compositions of the present invention can be administered with additional agent(s) in combination therapy, either jointly or separately, or by combining the pharmaceutical compositions and the additional agent(s) into one composition. The dosage is administered and adjusted to achieve maximal management of the conditions. For example, both the pharmaceutical compositions and the additional agent(s) are usually present at dosage levels of between about 10% and about 150%, more preferably, between about 10%) and about 80%>, of the dosage normally administered in a mono-therapy regimen.

EXAMPLES

[0177] The invention is further illustrated by the following examples, provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

Example 1: Solubility and hemolytic assay of sarcosine (SAR) substitution peptides Methods: Hemolytic Assay.

[0178] Peptides were diluted to 0.77 mM in factor B-depleted human sera (Complement Technologies, Inc.) and incubated for 1 hour at 37 °C. These peptides were then diluted with GVBS ⁺⁺ to equal 2.5% serum, of which 0.25 ml was combined with 0.4 ml of GVBS and 0.1 ml of sensitized sheep red blood cells (RBCs) and again incubated for 1 hour at 37 °C. The procedure was stopped by the addition of 4.0 ml of GVBS ^{~ ~} , centrifuged for 5 minutes at 1,620 x g, and the absorbance of the supernatants was read at 412 nm in a spectrophotometer. The percent lysis of each sample was standardized to that of the serum only control.

Results: Solubility of Sarcosine Substituted Peptides

[0179] TABLE 5 shows the solubility and hemolytic assay in factor B depleted serum of sarcosine (Sar) substitution peptides. The final concentration of peptide in factor B-depleted serum was 0.77 mM. Each peptide was evaluated in triplicate and the mean values are reported. Peptides not soluble in water were re-suspended in DMSO. In the hemolytic assay, soluble peptides are standardized to water and insoluble peptides standardized to DMSO.

TABLE 5

Peptide Name Solubility Hemolysis

Peptide Amino Acid Sequence SEQ ID NO

and Controls in water (%)

Water 100.00

GVBS ⁺⁺ - - 1.31

DMSO - - 95.13

PA IALILEPICCQERAA SSEEQQ IIDD NNOO :: : 33 NNoo 3.15

PA-IlSar (Sar) ALILEPICCQERAA SSEEQQ IIDD NNOO :: : 44 NNoo 84.49

PA-A2Sar I (Sar) LILEPICCQERAA SSEEQQ IIDD NNOO :: : 55 YYeess 37.72 PA-L3Sar IA (Sar) ILEPICCQERAA SSEEQQ IIDD NNOO :: : 66 YYeess 87.36 PA-I4Sar IAL (Sar) LEPICCQERAA SEQ ID NO : 7 Yes 93.19

PA-L5Sar IALI (Sar) EPICCQERAA SEQ ID NO : 8 Yes 85.13

PA-E6Sar IALIL (Sar) PICCQERAA SEQ ID NO : 9 No 5.19

PA-P7Sar IALILE (Sar) ICCQERAA SEQ ID NO: 10 Yes 3.01

PA-I8Sar IALILEP (Sar) CCQERAA SEQ ID NO: 11 Yes 33.92

PA-C9Sar IALILEPI (Sar) CQERAA SEQ ID NO: 12 Yes 5.63

PA-C10Sar IALILEPIC (Sar) QERAA SEQ ID NO: 13 No 56.18

PA-Ql l Sar ALILEPICC (Sar) ERAA SEQ ID NO: 14 No 3.02

PA-E12Sar IALILEPICCQ (Sar) RAA SEQ ID NO: 15 No 105.72

PA-R13Sar IALILEPICCQE (Sar) A SEQ ID NO: 16 No 3.42

PA-A14Sar IALILEPICCQER (Sar) A SEQ ID NO: 17 No 3.88

PA-A15Sar IALILEPICCQERA ( Sar ) SEQ ID NO: 18 No 21.68

[0180] Peptide derivatives PA-P7Sar (SEQ ID NO: 10) and PA-C9Sar (SEQ ID NO: 12) were both water soluble and inhibited complement activity to the same degree as the PA peptide (SEQ ID NO: 3). A dose-dependent inhibition of complement activity by both PA- P7Sar and PA-C9Sar in a hemolytic assays is shown in FIG. 1.

Example 2 - Solubility and hemolytic assay of PEGylated PA peptides.

Methods: Hemolytic Assay.

[0181] Polar Assortant peptides were serially diluted in undiluted factor B-depleted human sera (Complement Technologies, Inc.) and incubated for 1 hour at 37 °C. Water, GVBS ⁺⁺ and DMSO were included as controls. These peptides were then diluted with GVBS ⁺⁺ to equal 2.5% serum, of which 0.25 ml was combined with 0.4 ml of GVBS and 0.1 ml of sensitized sheep red blood cells (RBCs) and again incubated for 1 hour at 37 °C. The procedure was stopped by the addition of 4.0 ml of GVBS ^{~ ~} , centrifuged for 5 minutes at 1,620 x g, and the absorbance of the supernatants was read at 412 nm in a spectrophotometer. The percent lysis of each sample was standardized to that of the serum only control.

[0182] Initially, PA was PEGylated with 24 PEG moieties on the N terminus (dPEG24- PA, SEQ ID NO: 20), on the C terminus (PA-dPEG24, SEQ ID NO: 21), or on both the N terminus and C terminus (dPEG24-PA-dPEG24, SEQ ID NO: 19). All three PEGylated peptides were soluble in water and showed varying degrees of complement inhibition with PA-dPEG24 (SEQ ID NO: 21) inhibiting complement activation to the same degree as PA (FIG. 2A).

[0183] As shown in FIG. 2B, PA-dPEG24 (SEQ ID NO: 21) inhibited complement activation to the same degree as PA and displayed a broader dose-response most likely due to its increased solubility in aqueous solution. The structure of PA-dPEG24 (SEQ ID NO: 21) is shown in FIG. 3.

[0184] Next, PEGylated PICl derivatives were designed with decreasing numbers of PEG moieties on the C terminus. As shown in TABLE 6 below, most of these peptides retained solubility and complement inhibitory activity, with PA-dPEG24 (SEQ ID NO: 21) demonstrating the best inhibitory activity.

Results: Solubility of PEGylated Peptides

[0185] TABLE 6 shows the solubility and hemolytic assay in factor B depleted serum of PEGylated PA peptides. The final concentration of peptide in factor B-depleted serum was 0.77 mM. Peptides not soluble in water were re-suspended in DMSO. In the hemolytic assay, soluble peptides were standardized to water and insoluble peptides standardized to DMSO.

TABLE 6

SEQ ID Solubility

Peptide name Peptide sequence Hemolysis (%)

NO in water

Water - 100.00

DMSO - 95.17

PA-dPEG24 IALILEPICCQERAA-dPEG24 21 Yes 2.99

PA-dPEG20 IALILEPICCQERAA-dPEG20 22 Yes 11.41

PA-dPEG16 IALILEPICCQERAA-dPEG16 23 Yes 12.04

PA-dPEG12 IALILEPICCQERAA-dPEG12 24 Yes 11.41

PA-dPEG08 IALILEPICCQERAA-dPEG08 25 Yes 34.66

PA-dPEG06 IALILEPICCQERAA-dPEG06 26 No 44.82

PA-dPEG04 IALILEPICCQERAA-dPEG04 27 Yes 12.36

PA-dPEG03 IALILEPICCQERAA-dPEG03 28 Yes 12.57

PA-dPEG02 IALILEPICCQERAA-dPEG02 29 Yes 11.62 [0186] In vivo data in rats demonstrated that PA-dPEG24 (SEQ ID NO: 21) inhibits complement activation (as measured by red blood cell lysis in a hemolytic assay) by up to 90% within 30 seconds upon injection into rats with inhibitory activity still observed up to 4 hours (FIG. 20). The two doses were given intravenously (IV). This was compared to the vehicle control (normal saline) which shows maximal hemolysis at all time points. PA- dPEG24 (SEQ ID NO: 21) was effective in vivo in 0.9% NaCl + lOmM NaHP0 ₄ , but also works in saline and likely other common aqueous solutions (eg., Ringer's lactate, D5W, etc.) for IV infusion in humans.

Example 3 - Solubility and hemolytic assay of PEGylated and sarcosine (SAR) and/or alanine substitution peptides.

[0187] Table 7 shows the solubility and hemolytic assay in factor B depleted serum of PEGylated and sarcosine (SAR) substitution peptides. The final concentration of peptide in factor B-depleted serum was 0.77 mM. Peptides not soluble in water were re-suspended in DMSO. In the hemolytic assay, soluble peptides are standardized to water and insoluble peptides standardized to DMSO.

TABLE 7

Peptide name and SEQ ID Solubility Hemolysis

Peptide sequence

controls NO in water (%)

Water - - 100.00

DMSO - - 95.17

PA-dPEG24 IALILEPICCQERAA-dPEG24

21 Yes 2.99

PA-C9SarC10A IALILEPI (Sar) AQERAA

30 Yes 99.37

PA-C9SarD10 IALILEPI (Sar) QERAA

31 Yes 100.52

PA-P7SarC9Sar IALILE (Sar) I (Sar) CQERAA

32 Yes 82.72

PA-E6Sar-dPEG24 IALIL (Sar) PICCQERAA-dPEG24

33 Yes 12.88

PA-Ql l Sar-dPEG24 IALILEPICC (Sar) ERAA-dPEG24

34 Yes 12.04

PA-R13Sar-dPEG24 IALILEPICCQE (Sar) AA-dPEG24

35 Yes 4.26

PA-A14Sar-dPEG24 IALILEPICCQER (Sar) A-dPEG24

36 Yes 3.68

E6SarP7Sar IALIL (Sar) (Sar) ICCQERAA

37 Yes 3.57

E6SarC9Sar IALIL (Sar) PI (Sar) CQERAA

38 Yes 6.21

Ql l SarP7Sar IALILE (Sar) ICC (Sar) ERAA

39 Yes 3.22 Ql l SarC9Sar IALILEPI (Sar) C (Sar) ERAA

40 Yes 15.42

R13SarP7Sar IALILE (Sar) ICCQE (Sar) AA

41 No 26.35

R13SarC9Sar IALILEPI (Sar) CQE (Sar) AA

42 No 84.81

A14SarP7Sar IALILE (Sar) ICCQER ( Sar) A

43 Yes 4.37

A14SarC9Sar IALILEPI (Sar) CQER(Sar) A

44 No 14.73

E6AE12A-dPEG24 IALILAPICCQARAA-dPEG24

45 Yes 4.95

E6AE12AC9Sar IALILAPI (Sar) CQARAA

46 No 2.99

E6AE12AP7Sar IALILA(Sar) ICCQARAA

47 Yes 4.60

[0188] Changes were made to the PA peptide in which multiple amino acids were substituted with sarcosine and/or alanine. Some peptides were further combined with PEGylation. Multiple peptides retained the same level of inhibitory activity and solubility as PA-dPEG24 (SEQ ID NO: 21), including, PA-R13Sar-dPEG24 (SEQ ID NO:35), PA- A14Sar-dPEG24 (SEQ ID NO: 36), E6SarP7Sar (SEQ ID NO: 37), Ql l SarP7Sar (SEQ ID NO: 39), A14SarP7Sar (SEQ ID NO: 43), E6AE12A-dPEG24 (SEQ ID NO: 45), and E6AE12AP7Sar (SEQ ID NO: 47).

Example 4 -Minimum Inhibitory Concentration β ΙΟ) for PICl peptides Methods: Minimum Inhibitory Concentration (MIC) Assay.

[0189] A microdilution minimum inhibitory concentration (MIC) assay was performed where increasing amounts of PA-dPEG24 (SEQ ID NO :21) or sarcosine-derivatives (SEQ ID NO: 4-18, 30-47) was added to various bacteria growing in broth culture in a microtiter plate. The plates were then incubated at 37°C overnight and then the plates were read on a spectrophotometer at an absorbance of OD600 nm to determine the turbidity of the solution. Decreased turbidity is indicative of inhibition of bacterial growth.

Results

[0190] PA-dPEG24 (SEQ ID NO: 21) was able to dose-dependently inhibit

Staphylococcus aureus growth compared to a saline control (FIG. 4A) above 1 mM concentration and inhibited the same pathogen in a similar manner to the antibiotic vancomycin (FIG. 4B). PA-dPEG24 (SEQ ID NO: 21) was able to inhibit Klebsiella pneumonia at > 3 mg/ml similar to gentamycin (FIG. 4C) and also inhibit Pseudomonas aeruginosa (FIG. 4D) at > 13 mg/ml. The activity of PA-dPEG24 (SEQ ID NO: 21) was compared to 3 water soluble sarcosine substitution peptides (PA L3Sar (SEQ ID NO: 6), PA MSar (SEQ ID NO: 7) and PA L5Sar (SEQ ID NO: 8)) against Pseudomonas aeruginosa and it was found that all 3 sarcosine-derivatives peptides dose-dependently inhibited this species of bacteria at > 6 mg/ml (FIG. 4E). These results demonstrate a completely novel

antimicrobial property of these peptides which has not been previously reported that is completely independent of the complement suppressing properties of the PICl family of peptides.

[0191] Next, it was tested whether PA-dPEG24 (SEQ ID NO: 21), PA L3Sar (SEQ ID NO: 6), PA MSar (SEQ ID NO: 7) and PA L5Sar (SEQ ID NO: 8) could inhibit the growth of Gardnerella, a Gram variable, anaerobic coccobacillus that is a common cause of bacterial vaginosis. All 4 peptides inhibited the growth of Gardnerella (FIG. 5). It was also assayed whether PA-dPEG24 (SEQ ID NO: 21) could inhibit Gardner ella-mediated complement activation. In this assay normal human serum was incubated with Gardnerella in the presence of increasing amounts of PA-dPEG24 (SEQ ID NO: 21) for 1 hour at room temperature. Complement activation was assayed by measuring the production of C5a using ELISA methodology. Increasing amounts of PA-dPEG24 (SEQ ID NO: 21) inhibited C5a formation (FIG. 6). These results suggest that PICl peptides (e.g. SEQ ID NOs: 3-47) could be delivered locally in the vagina to treat bacterial vaginosis by inhibiting the replication of Gardnerella as well as Gardner ella-i ^' mtiated inflammation. The associated inflammation causes the symptoms of bacterial vaginosis and increases the risk of HIV-transmission by disrupting normal barrier defenses in the vagina.

Example 5 - Assay to Detect Complement-Mediated Hemolysis Can Discriminate

Erythrocyte Transfusions Likely to Cause Acute Transfusion Reactions (ATR)

Methods: Assay to Detect Complement-Mediated Hemolysis.

[0192] A 13 year old female B+ with sickle cell disease received three units of 0+ packed red blood cells (pRBCs) from two different donors. During the first transfusion of two units, the patient experienced hypotension (BP 89/39) followed by fever (Tmax 102.6°F). Transfusions of the third transfused product was tolerated without complications. Due to suspicion for an acute transfusion reaction (ATR), transfusion reaction work-up of the first transfused blood product revealed elevated anti-B titers. Subsequent investigative study of both identified donors indicated the following: Donor #1 was a 48 year old male with no reported transfusion adverse events. Donor #2 was a 61 year old female with prior pregnancies, but no reported transfusion adverse events.

[0193] Plasma from both donors were diluted with GVBS++ (veronal buffered saline with 0.1% gelatin, 0.15mM CaCl ₂ and ImM MgCl ₂ ) and incubated with purified erythrocytes from a B+ donor at 37°C for 1 hour. Free hemoglobin was measured by spectrophotometer at 412 nm. Classical complement activation was further evaluated by adding the classical complement pathway inhibitor PA-dPEG24 (SEQ ID NO: 21) to the hemolytic plasma sample prior to incubation with erythrocytes.

Results

[0194] Complement mediated hemolysis was >100-fold different between the two plasma samples from the different donors (TABLE 8). TABLE 8 shows plasma anti-B titers and complement hemolysis for two 0+ donors. Classical complement activation was confirmed by blocking hemolysis with the classical complement pathway inhibitor, PA-dPEG24 (SEQ ID NO: 21).

TABLE 8

Anti-B titer (plasma) Complement hemolysis Hemolysis in RT 37°C IgG (Abs 412nm) presence of PA- dPEG24

Donor 0+ #1 128 64 256 0.747 0.008 Donor 0+ #2 128 16 256 0.007

[0195] This data demonstrates that PICl peptides (e.g. SEQ ID NOs: 3-47) could be utilized to ascertain if ATRs are complement mediated. The data in TABLE 8 above is shown in FIG. 7 along with a dose-response PA-dPEG24(SEQ ID NO: 21) inhibition study of the highly hemolytic plasma sample (FIG. 8). A dose-response inhibition of hemolysis was demonstrated with PA-dPEG24 (SEQ ID NO: 21) up to >95% inhibition. This showed that the type-0 RBC transfusion leading to ATR in the type-B recipient was a classical complement-mediated event. FIG. 8 showed it was possible to pharmacologically block the extremely robust hemolytic activity mediated by this donor's plasma in this ATR in vitro model.

Conclusion

[0196] Past methodologies have not been able to adequately predict the likelihood of complement-mediated ATR. In this case two units of type-0 RBCs were given to a type-B recipient who then suffered ATR. There was estimated to be about 30 - 40% plasma by volume in a unit of packed RBCs (pRBCs) (i.e. 150 - 200ml plasma per 500ml unit). The antibodies in the plasma of the RBC transfusions likely initiated classical pathway complement-mediated ATR. The IgG titers for the plasma from donor of each RBC transfusion were both high, and thus non-predictive of this platelet transfusion caused the AHTR. A CH50-type complement hemolytic assay, however, was able to readily identify which type-0 plasma causes massive hemolysis of type-B erythrocytes (>100-fold increase). Therefore, a hemolytic complement assay performed before platelet transfusion would have identified that one of the units of platelets was likely to cause AHTR and could have prevented this serious adverse event.

[0197] Therefore, PICl peptides (SEQ ID NOS: 3-47) can be used to discriminate risk of complement-mediated ATR before a blood transfusion. This data also supports the use of PICl peptides (SEQ ID NOS: 3-47) as a treatment for ATRs in various clinical scenarios.

Example 6 - PICl (SEQ ID NO: 21) Enhances Survival of Human Erythrocytes in an Animal Model of Acute Intravascular Hemolytic Transfusion Reaction

Methods: Ethics Statement.

[0198] Adolescent male Wistar rats (200-250g) with indwelling jugular catheters were purchased from Harlan laboratories and used under the Eastern Virginia Medical School (EVMS) IACUC (Institutional Animal Care and Use Committee) approved protocols 12-003 and 15-003. Central line maintenance was followed as recommended by the animal vendor, with approval from the EVMS IACUC.

[0199] After written informed consent was provided, a healthy human volunteer donated AB blood which was used to generate purified red blood cells (RBCs) (EVMS IRB protocol 02-06-EX 0216). These RBCs were also used for in vitro analyses as the source of human RBCs (huRBC).

Methods: Human RBC Purification and Animal Experiments

[0200] Human RBCs (huRBCs) acquired the morning of the animal experiments were processed as described in (Shah et al., "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis." Transfusion. 2014). This process generates 1ml huRBCs at 80% hematocrit, which was given as a 15% transfusion to the rats. [0201] The AIHTR Wistar rat model was previously established in which cobra venom factor (CVF) was demonstrated to inhibit complement-mediated lysis of huRBCs, whereas animals receiving normal saline (NS) demonstrated classical pathway, complement-mediated hemolysis. (Shah TA, Mauriello CT, Hair PS, et al. "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis." Transfusion. 2014). Dosing of the PEGylated derivative, PA-dPEG24 (SEQ ID NO: 21) (New England Peptide, MA) was established in Wistar rats. FIG. 21 A shows the sequence of events in a dosing experiment using a low (20mg) and high dose (40mg) of PA-dPEG24 (SEQ ID NO: 21) in the AIHTR animal model. Animals were randomly assigned to various groups where they received either normal saline (n=6), 20mg PA-dPEG24 (SEQ ID NO: 21) (n=4) or 40mg PA-dPEG24 (SEQ ID NO: 21) (n=7) before the huRBC transfusion (prophylaxis arm). FIG. 2 IB shows the second set of experiments where one group of animals received 40mg PA- dPEG24 (SEQ ID NO: 21) (n=8) before the huRBC transfusion (prophylaxis arm) and a separate set of animals (n=10) receiving 40mg PA-dPEG24 (SEQ ID NO: 21) after the huRBC transfusion (rescue arm). For both experiments, animals allotted to the saline control group received NS prior to huRBC transfusion whereas the CVF group received 130μg CVF (Complement Technologies, Inc.) intra-peritoneum (LP.) 24 hours prior to the huRBC transfusion. In an additional experiment, separate groups of animals were injected with 40mg Immune globulin intravenous (POLYGAM®, Baxter Healthcare Corporation, CA) IVIG (n=3) and 40mg PA-dPEG24 (SEQ ID NO: 21) (n=3) before the huRBC transfusion (IVIG vs. PA-dPEG24).

[0202] Using weight-based doses of ketamine and acepromazine, animals were sedated throughout the course of the experiment with periodic monitoring of vital signs. Blood samples were collected into EDTA microtainer tubes (BD) from the animals prior to the huRBC transfusion and then at 30 seconds, 5 minutes, 20 minutes, 60 minutes and 120 minutes after the huRBC transfusion. In the group of animals receiving IVIG vs. PA- dPEG24 (SEQ ID NO: 21), blood samples were drawn prior to the huRBC transfusion and then 30 seconds, 5 minutes , 20 minutes, 60 minutes ,120 minutes, 180 minutes, 240 minutes, 300 minutes and 360 minutes after the huRBC transfusion. These samples were kept shaking at room temperature prior to processing and centrifuged at 2655xg for five minutes to separate out the plasma and sediment the RBCs. Plasma was aliquoted and the RBC pellet was processed separately as described below. Upon completion of the final (120 minutes or 360 minutes) blood draw, the animal was euthanized using Fatal Plus (Vortech). A necropsy was completed to collect organs (liver, spleen and bilateral kidney) for histopathology. In the experiment IVIG vs. PA-dPEG24 (SEQ ID NO: 21), bilateral kidneys acquired from each animal were weighted separately and stored in formalin prior to processing and paraffin embedding. Hematoxylin and eosin (H&E) stained sections were reviewed by a blinded pathologist.

Methods: Flow Cytometry

[0203] Flow cytometry was performed on the collected RBCs using a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) with DXP 8 Color 488/637/407 Upgrade (Cytek Development, Fremont, CA). Samples were stained with either FITC and/or APC labeled antibodies. The data was acquired using Cytek FlowJo CE version 7.5.110.6.

Approximately lxlO ⁵ and 5xl0 ⁵ events per sample were gathered for single and double labeled flow, respectively. Data was analyzed using FlowJo X version 10.0.7r2 (FlowJo LLC, Ashland, OR). Although FITC and APC are not expected to cause much spillover, digital compensation was used in the analysis.

[0204] Single labeled flow: The RBC pellet collected after separating the plasma was washed, diluted and stained with FITC-conjugated anti-human CD235a (glycophorin A, eBioscience, San Diego, CA) at 1 :200 in GVBS - - (veronal -buffered saline (VBS) with 0.1% gelatin, 0.01 mol/L EDTA) over 20min while shaking at room temperature to minimize agglutination. An antibody control consisted of the RBC pellet washed, diluted and unstained or stained with mouse IgG2b Iso Control FITC at 1 :200 (eBioscience, San Diego, CA).

[0205] Dual labeled flow: The RBC pellet generated above was washed and double stained with APC-conjugated mouse anti-human CD235a (glycophorin A, BD Bioscience, San Jose, CA) at 1 :50 and FITC-conjugated goat-anti-rat C3 (MP Biomedical, Santa Ana, CA) at 1 :200 in GVBS - - for 20min at room temperature. Three controls consisting of unstained RBCs, RBCs acquired at 30sec stained with the APC tagged antibody and FITC tagged antibody were used for quadrant analysis.

[0206] In vitro flow cytometry experiment: 100% complement sufficient Wistar rat sera (Innovative Research, Novi, MI) was incubated with PA-dPEG24 (SEQ ID NO: 21) at 12mg/ml (4 mMol) for 5 minutes at room temperature to generate PA-dPEG24 (SEQ ID NO: 21) treated serum. HuRBC's prepared as above were then incubated with PA-dPEG24 (SEQ ID NO: 21) treated serum or with 100% complement sufficient Wistar rat sera untreated serum for 5 min each at 37°C. The cells were then washed twice with GVBS to terminate complement activation. These cells were then double stained with APC-conjugated mouse anti-CD235a and FITC-conjugated goat anti-C3 as described above. After washing, the cells were combined with 85% non-opsonized, non-stained cells to mimic the 15% transfusion used in vivo and analyzed by flow cytometry using the same conditions stated in dual labeled flow, above.

Methods: Hemoglobin and Bilirubin Measurements

[0207] Plasma generated from the above experiments was analyzed for the free hemoglobin using spectrophotometry, as described previously (Shah et al. "Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway." J Transl Med 2014, 12. For bilirubin measurements, the pre-bleed and 120 minute plasma samples from the prophylaxis (n=8) and NS (n=7) groups were analyzed for the amount of bilirubin present using the Bilirubin Assay Kit (Sigma- Aldrich, St. Louis, MO) in half the manufacturer's recommend volume. Due to large amounts of hemolysis in the latter time points and the associated optical interference in bilirubin analysis, all the samples were pre-treated with HemogloBindTM (Biotech, NJ) prior to analysis with the Bilirubin Assay Kit. (Koseoglu M, Hur A, Atay A, Cuhadar S. "Effects of hemolysis interferences on routine biochemistry parameters." Biochemia medica. 201 l;21(l):79-85].

Results

[0208] PA-dPEG24 (SEQ ID NO: 21) blocked AIHTR in rats when given

prophylactically before the transfusion, as well as when PA-dPEG24 (SEQ ID NO: 21) was administered as rescue treatment after transfusion. PA-dPEG24 (SEQ ID NO: 21) was compared in a dose comparable fashion against intravenous immunoglobulin (IVIG) in the AIHTR disease animal model. IVIG is being used in clinical practice in conjunction with phototherapy to reduce the need for exchange transfusion in neonates with

hyperbilirubinemia due to ABO incompatibility related hemolytic disease [Schwartz HP, Haberman BE, Ruddy RM. "Hyperbilirubinemia: current guidelines and emerging therapies." Pediatric emergency care. 2011;27(9):884-889; "Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation." Pediatrics. 2004;114(l):297-316; Cortey A, Elzaabi M, Waegemans T, Roch B, Aujard Y. "Efficacy and safety of intravenous immunoglobulins in the management of neonatal hyperbilirubinemia due to ABO

incompatibility: a meta-analysis." Archives de pediatric : organe officiel de la Societe francaise de pediatric 2014;21(9):976-983]. Example 7 - PICl (SEQ ID NO: 21) Enhances Survival of Human Erythrocytes in an Animal Model of Acute Intravascular Hemolytic Transfusion Reaction - Prophylactic administration of SEQ ID NO: 21 reduces hemolysis of huRBCs in the AIHTR model

[0209] Previous work had established the AIHTR model in Wistar rats and demonstrated that a 15% transfusion of HuRBC resulted in classical pathway-mediated intravascular lysis of the xenotransfused cells [Shah et al. "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis " Transfusion. 2014

Nov;54(l l):2892-900. ] "Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway." J TranslMed 2014; 12: 181.]. Depletion of complement activity by CVF led to increased survival of HuRBC as shown by flow cytometry and free hemoglobin measurements with the transfused cells eventually being sequestered in the spleen and liver [Shah et al. "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis " Transfusion. 2014 Nov;54(l l):2892-900.]. To assess the efficacy of PA-dPEG24 (SEQ ID NO: 21) inhibition on the survival of huRBCs in this AIHTR model, animals were prophylactically treated with PA-dPEG24 (SEQ ID NO: 21) at doses of 20 and 40 mg/animal followed by 15% xenotransfusion of HuRBCs and blood draws at the indicated time points (FIG. 22A). Animals pre-treated with CVF served as a positive control for complement depletion and a separate set of animals received NS (vehicle control). Plasma isolated from NS-treated animals demonstrated a spike of hemolysis as assayed by released free hemoglobin (FIG. 22A). In contrast, animals pre-treated with CVF had low levels of free hemoglobin as previously reported (FIG. 22A) [Shah et al. "Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis " Transfusion. 2014 Nov;54(l l):2892-900.]. Animals receiving both doses of PA-dPEG24 (SEQ ID NO: 21) showed decreased levels of free hemoglobin similar to that observed for CVF-treated animals; animals receiving 40mg PA-dPEG24 (SEQ ID NO: 21) showed significant reduction of free hemoglobin compared to NS control animals at 5 minutes and 20 minutes (P<0.05) (FIG. 22A). To verify the complement-mediated destruction of the transfused huRBC, RBCs from blood collected at the indicated time points were isolated and labeled with antibody to human glycophorin A (CD235a) and analyzed by flow cytometry. Animals receiving 20 or 40 mg doses of PA-dPEG24 (SEQ ID NO: 21) showed survival of transfused cells comparable to that observed in the CVF treated animals up to 20 minutes (FIG. 22B). There was no statistically significant difference observed between the effects caused by both doses of PA-dPEG24 (SEQ ID NO: 21) and CVF in these experiments. These results show the protective effect of PA-dPEG24 (SEQ ID NO: 21) on the survival of incompatible HuRBCs in the animal model of AIHTR.

Example 8 PICl (SEQ ID NO: 21) Enhanced Survival of Human Erythrocytes in an Animal Model of Acute Intravascular Hemolytic Transfusion Reaction - Administration of SEQ ID NO: 21 after HuRBC transfusion protects the transfused cells from complement-mediated hemolysis

[0210] PA-dPEG24 (SEQ ID NO: 21) was able to show protection of the HuRBC when administered to rats prior to xenotransfusion, It was also discovered that PA-dPEG24 (SEQ ID NO: 21) given immediately after xenotransfusion could protect the HuRBCs from complement-mediated attack. Rats received a 40mg dose of PA-dPEG24 (SEQ ID NO: 21) either 30 seconds before (prophylaxis arm) or 30 seconds after xenotransfusion of HuRBCs (treatment /rescue arm). A separate group of animals also received NS vehicle control or were pre-treated with CVF (FIG. 2 IB). The protective effect of 40 mg PA-dPEG24 (SEQ ID NO: 21) given before the transfusion (prophylaxis group) and after the transfusion (treatment /rescue group) on the amount of hemolysis as assayed by free hemoglobin was significantly reduced compared to the animals who received NS at 5 minutes (P<0.005) and 20 minutes (P<0.005) (FIG. 23 A), representing the greatest amount of hemolysis in this AIHTR model (FIG. 22A & 23 A) FIG. 23B shows the cumulative hemolysis occurring in the various groups of animals (Saline, Prophylaxis, Treatment and CVF). Both the treatment and prophylaxis groups demonstrated reduced hemolysis compared to the saline group of animals (P<=0.05). Another common pathway of hemoglobin metabolism when it is released into the plasma as a result of intravascular hemolysis is indirect bilirubinemia [Strobel E. "Hemolytic Transfusion Reactions." Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie . 2008;35(5):346- 353]. NS treated animals showed a large increase in free bilirubin between the pre-bleed and 120 minute time point (FIG. 23C), consistent with RBC lysis as assessed by free hemoglobin and flow cytometry. In contrast to the NS treated animals, 40mg PA-dPEG24 (SEQ ID NO: 21) given prior to the transfusion of HuRBCs significantly reduced the amount of bilirubin circulating at 120 minutes compared to the animals receiving NS (P=0.0015) (FIG. 23C). Example 9 - Peptide Inhibitor of Complement CI (¾EQ ID NO: 21) Enhances Survival of Human Erythrocytes in an Animal Model of Acute Intravascular Hemolytic Transfusion Reaction - Assessment of complement deposition on transfused HuRBCs

[0211] The results described above demonstrate that PICl (SEQ ID NO: 21) can be utilized in both a prophylactic and treatment strategy to prevent hemolysis of transfused huRBC as assayed by huRBC survival, free hemoglobin levels and bilirubin release. To gain a more complete understanding of the role of complement deposition on the transfused cells, an analysis of C3 binding to huRBCs was conducted. To establish experimental conditions, an in vitro study was performed using HuRBCs exposed to rat serum followed by analysis of C3 deposition on the cells by flow cytometry. Rat serum treated with and without PA- dPEG24 (SEQ ID NO: 21) was added to HuRBCs and allowed to opsonize for 5 minutes. Cells were then analyzed by dual labeled flow cytometry with representative plots for HuRBCs exposed to untreated or PA-dPEG24 (SEQ ID NO: 21) -treated serum shown in FIG. 24A and 24B, respectively. Quantification of HuRBCs opsonized in rat serum with and without PA-dPEG24 (SEQ ID NO: 21) showed a relative increase in the number of cells with no C3 deposition in PA-dPEG24 (SEQ ID NO: 21)-treated serum (FIG. 24C). When the fraction of dual labeled cells relative to the total number of glycophorin A labeled cells was calculated, there was a >1.5 fold reduction in C3 deposition on cells incubated with PA- dPEG24 (SEQ ID NO: 21)-treated serum versus untreated serum (FIG. 24D). In untreated serum, a larger population of dual labeled cells (Q2) was observed to shift over to Quadrant 1 which represented cells with C3 deposition. These findings demonstrated that PA-dPEG24 (SEQ ID NO: 21) rescued HuRBCs from opsonization and impending hemolysis through activation of the complement proteins in vitro.

[0212] To evaluate the effect of PA-dPEG24 (SEQ ID NO: 21) on C3 deposition of transfused HuRBCs in the AIHTR model, RBCs isolated from the blood of animals that received either CVF, NS or PA-dPEG24 (SEQ ID NO: 21) prior to xenotransfusion or post- xenotransfusion were subject to two color flow cytometry for both human glycophorin A expression and rat C3. Representative flow cytometry plots of RBCs at 30 seconds, 5 minutes and 20 minutes post-transfusion are shown for NS (FIG. 25 A-C) and PA-dPEG24 (SEQ ID NO: 21) treated (FIG. 25D-F) animals. NS-treated animals exhibited an increased number of double-positive cells over time compared to PA-dPEG24 (SEQ ID NO: 21)-treated animals. Conversely, PA-dPEG24 (SEQ ID NO: 21)-treated animals had more glycophorin A positive cells consistent with increased RBC survival as assessed by single label flow cytometry and free hemoglobin measurements (FIGS. 22A-B & 23A-C). To more precisely quantify these results, the numbers of events captured by flow cytometry were analyzed and graphed (FIG. 26A). At 30 seconds after HuRBC transfusion prophylactic treatment with PA-dPEG24 (SEQ ID NO: 21) showed a dramatic increase in the numbers of circulating HuRBCs (Human - No C3) compared with no intervention (NS) or even CVF-treated animals (FIG. 26A). The amount of surviving HuRBCs at 30 seconds for the PA-dPEG24 (SEQ ID NO: 21) treatment group was similar to the animals receiving NS, which was expected as PA- dPEG24 (SEQ ID NO: 21) was given 30 seconds after the transfusion (FIG. 26A). At 5 and 20 minutes after transfusion, groups of animals receiving prophylaxis and treatment with PA- dPEG24 (SEQ ID NO: 21) showed decreased C3 deposition on the surface of circulating HuRBCs compared with animals receiving no intervention (NS) (FIG. 26B). For animals receiving PA-dPEG24 (SEQ ID NO: 21) after transfusion (treatment arm), while many HuRBCs were cleared from the circulation prior to PA-dPEG24 (SEQ ID NO: 21) treatment (FIG. 26A), HuRBCs persisted in circulation through 20 minutes at higher numbers than that observed for the NS group (FIG. 26C). In order to evaluate complement attack of innocent bystander RBCs (i.e. rat RBCs), C3-fragment bound RBCs that were not labeled with anti- glycophorin A were counted. Prophylactic administration of PA-dPEG24 (SEQ ID NO: 21) decreased innocent bystander attack of non-glycophorin A RBCs at all observed time points compared to the NS control animals. Post-transfusion treatment with PA-dPEG24 (SEQ ID NO: 21) decreased 'innocent bystander' attack at the 5 and 20 minute time points (FIG. 26D).

Example 10 - Investigation of Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid - Complement Anaphylatoxins in CF Lung Fluid

Methods: Ethics Statement.

[0213] Sputum samples were obtained from consented patients as part of their standard of care visit at the Children's Hospital of The King's Daughters Cystic Fibrosis Center and collected from the Clinical Microbiology Laboratory prior to being discarded. This was performed under an Eastern Virginia Medical School IRB approved protocol 12-08-EX-0200. Samples were given a numerical code that was linked in an encrypted file to the clinical database. Control sputum samples were obtained from healthy human volunteers.

Methods: Sputum Sols

[0214] Sputum samples were obtained as expectorated sputum and placed immediately on ice. To confirm that induction of sputum did not alter complement in the sol, a control experiment was performed with a healthy human volunteer who produced an expectorated sputum and an induced sputum showing no differences in complement activation or complement effector concentrations. The soluble (sol) fraction was generated by cold (4°C) centrifugation at 14,000 g for 60 minutes and recovery of the free flowing liquid fraction, similar to methods previously described [Davies JR, Svitacheva N, Lannefors L, et al.

"Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions." Biochem J 1999;344 Pt 2:321-30]. The sols were aliquoted and frozen at - 80°C.

Clinical Data

[0215] Clinical Data were obtained from data entered into Port CF for the clinic visit at which the sputum sample was collected and from review of the medical record. The FEV1% predicted value was obtained from pulmonary function testing performed at the clinic visit when the sputum sample was collected. The bronchiectasis score was based on the most recent radiographic lung study prior to obtaining the sputum sample, usually plain

radiograph. A bronchiectasis score was assigned as follows: 0 = normal; 1 = 1 lobe, mild; 2 = 2-4 lobes; 3 = all lobes. Cystic fibrosis related diabetes (CFRD) status was based on the most recent endocrinology assessment prior to obtaining the sputum sample. CFRD status was scored as follows: 0 = normal; 1 = glucose intolerance; 2 = CFRD. Part of each sputum sample was sent for microbiologic testing in the clinical microbiology laboratory at CUKD and organisms were recorded. The organisms were categorized as to whether the following were present or absent: Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex, or Candida species. Patient medications at the time of clinic visit were also recorded. Medications were categorized as to whether the following were present or absent: systemic corticosteroid, inhaled corticosteroid, azithomycin, inhaled antibiotic, or systemic antibiotic (excluding azithromycin).

Methods: ELISAs & Western Blots

[0216] The C5a concentration in sputum sol was quantitated using a C5a ELISA kit (R&D Systems). C3a and C4a from sputum sol were both measured using their respective ELISA kits (BD Biosciences). Bound C3-fragments were determined using a total C3 ELISA. Briefly, a goat anti-human C3 antibody (Complement Technology) was used to coat flat-bottom Immulon-2 plates the night before. The plates were then washed with PBST (PBS with 0.1% TWEEN), blocked for two hours with 3% BSA/PBS, washed again, and followed by the addition of samples and a pure C3 standard (Complement Technology) for 1 hour in block buffer. Plates were washed, incubated with a chicken anti-human C3 antibody (Sigma) for 1 hour, washed again, and incubated with a goat anti-chicken HRP antibody (Genway). Plates were developed with TMB Substrate Solution (Thermo Scientific) and stopped with 2.5 N H ₂ S0 ₄ (Hair PS, Echague CG, Rohn RD, et al. "Hyperglycemic conditions inhibit C3 -mediated immunologic control of Staphylococcus aureus." J Transl Med 2012;10:35]. Bound C4-fragments were evaluated with the same ELISA method as for C3, except using a goat anti -human C4 antibody to coat the plates, a pure C4 as the standard (both Complement Technologies), and a chicken anti-human C4 antibody (Abeam) as the primary antibody.

[0217] C5a fragments were analyzed by Western Blot using a mouse anti-human C5a antibody (R&D Systems) to probe followed by a goat anti-mouse HRP antibody (Sigma) and detected with ECL.

Results

[0218] To evaluate whether complement anaphylatoxins were elevated in CF lung fluid, CF sols from the sputum of CF patients were assayed for each complement anaphylatoxin and compared with sputum sols from healthy human controls. The most inflammatory complement anaphylatoxin was C5a, which was assayed by ELISA and Western blot. Mean C5a concentration in CF sols was 4.8-fold higher (P = 0.04) compared with the mean for healthy controls (FIG. 10A). Qualitative analysis by Western blot probing for C5a confirmed that large amounts of C5a were present in CF sols compared with controls (FIG. 10B). C3a is a complement effector that is generated during activation of the central complement component C3. Mean C3a concentrations in CF sols was 4-fold higher (P = 0.03) compared with controls (FIG. IOC). C4a is the least potent of complement anaphylatoxins and is generated during classical or lectin pathway complement activation. Mean C4a concentration was 2-fold higher in CF sols (P = 0.05) compared with controls (FIG. 10D). Together these data show that the concentration of complement anaphylatoxins in CF lung fluid was significantly elevated, suggesting significant complement activation in CF lung fluid. The potent ability of C5a to recruit neutrophils and stimulate degranulation [Lambris JD, Sahu A, Wetsel RA. "The chemistry and biology of C3, C4, and C5. In: Volanakis ΓΕ, Frank MM, (eds)." The human complement system in health and disease. New York: Marcel Dekker; 1998, 83 -118; Mollnes TE, Brekke OL, Fung M, et al. "Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation." Blood 2002; 100(5): 1869-77; Laursen NS, Magnani F, Gottfredsen RH, et al. "Structure, function and control of complement C5 and its proteolytic fragments." Curr Mol e<f 2012; 12(8): 1083-97], suggested that C5a could contribute to the high concentrations of neutrophil elastase in CF lung fluid, which is associated with parenchymal destruction. Additionally, the elevated levels of C4a in CF lung fluid, suggested that much of the complement activation occurring in CF lung fluid may be occurring via the classical or lectin complement pathways.

Example 11 - Investigation of Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid - Complement Opsonization of Staphylococcus aureus in CF Lung Fluid

Methods: S.aureus Opsonization With CF Sols

[0219] Staphlycoccus aureus strain Reynolds was grown in 2% NaCl Columbia broth at 37°C to log phase, washed twice with GVBS ⁺⁺ (vernal buffered saline with 0.1% gelatin, 0.15 mM CaCl ₂ , and 1.0 mM MgCl ₂ ), and resuspended to 1 ^χ 10 ⁹ cells/ml. An equal volume of bacteria and CF or control sol were incubated for one hour at 37°C. The bacteria were washed twice with GVBS ^{" "} (VBS with 0.1% gelatin and 0.01 M EDTA), and then stripped of bound complement fragments using methylamine, as previously described [Cunnion KM, Lee JC, Frank MM. "Capsule production and growth phase influence binding of complement to Staphylococcus aureus." Infect Immun 2001;69(11): 6796-803 ]. Results

[0220] In order to evaluate whether complement in CF lung fluid could adequately opsonize a pathogenic bacteria, CF sol opsonization of Staphylococcus aureus was evaluated. Given the large amounts of complement activation that had already occurred in the CF lung fluids, it was important to determine whether there was residual complement-mediated host defense. Staphylococcus aureus were incubated with CF or control sols, washed and stripped of bound C3 -fragments and bound C4-fragments. Staphylococcus aureus was robustly opsonized in CF sol yielding a nearly identical mean level of bound C3 -fragments compared with normal controls (FIG. 11 A). Staphylococcus aureus was also robustly bound by C4- fragments (P = 0.13) with a non-significant trend towards increased mean C4-fragment binding by CF sols compared with normal controls (FIG. 1 IB). These results suggested that CF lung fluid retained a normal capacity to opsonize bacteria, suggesting that this facet of host defenses is not compromised. These results also show that despite significant complement activation having occurred, as evident by very high anaphylatoxin levels, significant complement remains in CF lung fluid. This suggested a cycle of complement activation and repletion consistent with persistent inflammation. The robust opsonization with C4-fragments suggested that the classical or lectin complement pathway was active in CF lung fluid and may be the predominant pathway of complement activation.

Example 12 - Investigation of Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid - C5a generation in CF lung fluid by Pseudomonas aeruginosa and Staphylococcus aureus

[0221] Pseudomonas aeruginosa was acquired as a discarded de-identified clinical isolate. It was grown in Mueller Hinton broth to log phase and washed and re-suspended as was done with the Staphylococcus aureus. Both bacteria were gently heat killed by incubating in a 70°C water bath for 15 minutes. Samples were plated to confirm that the bacteria were dead. CF or control sols were incubated with either live or dead Pseudomonas aeruginosa or Staphylococcus aureus at equal volumes for one hour at 37°C. Afterward, the samples were spun at 14,000 ^χ g ior 5 minutes and the supernatant was collected and analyzed for C5a. Equal volume of CF sol and PA-dPEG24 (SEQ ID NO: 21) [Shah et al. "Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway. " J TranslMed20\A; \2: \%\; Mauriello CT, Pallera HK, Sharp JA, et al. "A novel peptide inhibitor of classical and lectin complement activation including ABO incompatibility." Mol Immunol 2012;53(l-2): 132-9], at 50 mg/ml, or saline for controls, were combined and pre-incubated for 30 minutes at room temperature. Afterward, 5 x 10 ⁷ CFU heat-killed Pseudomonas aeruginosa were added and incubated at 37° C for 30 minutes. The samples were then spun at 14,000 ^χ g for 2 minutes and the supernatant was collected and assayed in the C5a ELISA.

[0222] In order to evaluate whether CF lung fluid challenged with pathogenic bacteria commonly present in CF lungs would generate new C5a, the most inflammatory

anaphylatoxin, CF sols were incubated with live and dead Pseudomonas aeruginosa and Staphylococcus aureus. Live and dead versions of each bacteria were tested because both forms are likely to be present in an infected CF lung. Additionally, secreted factors or adaptive changes that could be produced by live bacteria would also alter C5a generation. Before and after incubation with bacteria, C5a concentrations were measured in CF to determine if new C5a were generated. Incubation of CF sols with live or dead Pseudomonas aeruginosa lead to an average increase in C5a generation of 2.3-fold (P = 0.02). Similarly, incubation with live or dead Staphylococcus aureus lead to an average increase in C5a generation of 2.4-fold (P = 0.02) (FIG. 12A). These data also showed a difference in C5a generation between live or dead Pseudomonas aeruginosa (P = 0.05), but not between live or dead Staphylococcus aureus. This relationship between live or dead Pseudomonas aeruginosa challenge appeared consistent for the different CF sol samples (FIG. 12B), but there was no consistent relationship between live or dead Staphylococcus aureus challenge (FIG. 12C). These data showed that Pseudomonas aeruginosa and Staphylococcus aureus both provoke robust generation of the highly inflammatory C5a anaphylatoxin in CF lung fluid, suggesting that the presence of these bacteria in the CF lung may be enhancing inflammation and subsequent host tissue damage. Pseudomonas aeruginosa may have some ability to moderate C5a generation in CF lung fluid compared to dead Pseudomonas aeruginosa.

[0223] In order to evaluate the likely complement pathways by which Pseudomonas aeruginosa was activating C5a generation, an inhibitor of classical and lectin complement pathway activation was tested. Peptide inhibitors of complement CI are small peptide inhibitors of the classical and lectin pathways of complement activation CF sols were incubated in buffer alone, with dead Pseudomonas aeruginosa, or with PA-dPEG24 (SEQ ID NO: 21) and dead Pseudomonas aeruginosa (FIG. 12D). Dead Pseudomonas aeruginosa increased C5a concentration by 1.6-fold compared to incubation of the CF sol in buffer alone. Addition of PA-dPEG24 (SEQ ID NO: 21) to the CF sol decreased C5a generation by Pseudomonas aeruginosa (P = 0.001) to a level not significantly different from CF sol alone (P = 0.22). Thus, addition of a classical/lectin pathway inhibitor blocked C5a generation by Pseudomonas aeruginosa, suggesting that Pseudomonas aeruginosa activates complement and C5a generation via the classical or lectin complement pathways. This finding was congruent with elevated C4a levels and robust C4-fragment opsonization.

Example 13 - Investigation of Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid - Complement anaphylatoxin correlation with clinical characteristics

Methods: Statistical Analysis of Clinical Measures With C5a/C3a and of Laboratory Data

[0224] The data were analyzed using SAS 9.4 (SAS Institute, Cary, NC) and SPSS 19 (SPSS Inc., Chicago, IL) software. The level of significance was set at 0.05. Pearson and Spearman correlation coefficients were reported, where appropriate, for C5a level, C3a level, and the clinical measures. Descriptive statistics were reported for C5a and C3a level stratified by clinical measures. Simple linear regression and Mann-Whitney U tests were used, where appropriate, to determine associations between C5a level, C3a level, and the clinical measures. A multivariable linear regression model for FEV1% was used to determine associations with C5a level and C3a level.

[0225] Medians, quartiles, and 90 ^th percentiles were calculated using PSI plot. Means and standard error of the means (SEM) were calculated from independent experiments. Statistical comparisons were made using Student's t-test where appropriate with a level of significance set at 0.05.

Results

[0226] CF sputum samples were collected from 15 patients. The clinical characteristics of these 15 subjects is shown in TABLE 9. The subjects span a wide range of ages from 2 - 65 years old with a median age of 19. Median FEV1% predicted was 59; for children (n = 7) the median BMI was 48% and for adults (n = 4) the median BMI was 23.87 mg/kg ² . Clinical data collected at the time of sputum sampling was obtained for a wide range of measures including FEV1% predicted, BMI percentage (children), bronchiectasis, CFRD status, pathogenic microorganisms cultured from the sputum and medications (i.e. corticosteroids, azithromycin, and antibiotics). Statistical correlations were assessed between the

anaphylatoxins, C5a and C3a, and the clinical measures, see TABLE 10. Increased concentration of the highly inflammatory C5a positively correlated with increased age (r = 0.53, P = 0.04), as shown in FIG. 13A. Increased C5a concentration correlated inversely with BMI percentile in children (r = -0.77, P = 0.04) as shown in FIG. 13B. Increased C3a levels positively correlated with increased FEV1% predicted (r _s = 0.63, P = 0.02), as shown in FIG. 13C. Microorganisms (i.e. Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus, or yeast) recovered from the sputum, corticosteroids, azithromycin, or antibiotics did not correlate with levels of C5a or C3a. These results showed that increasing C5a

concentration correlated with decreased BMI percentile in children, suggesting that increased complement inflammatory C5a in lung fluid may be associated with poorer overall health in children with CF. C3a level positively correlated with FEV1% predicted, suggesting a potentially protective effect from C3a on CF lung function. TABLE 9 - Characteristics of the CF subjects.

Median (range)

Age. v 19(2-65)

Gender, % 60

female

Child BMI. % 26(10-70)

Adult BMI 23.8 (21.8-25.9)

1 KM"., 59 (23 - 99)

TABLE 10 - Correlation coefficients for complement effectors and clinical measures

C5a ng/ml) C3a (ng/ml)

P value (H ₀ : rho=0) Child BMI, % 0.04 0.38

N 7 7

Bronchiectasis

P value (H ₀ : rho=0) 0.68 0.61

Score

N 15 14

Pearson correlation coefficient

Spearman correlation coefficient

Example 14 -Modulation of Clq Interaction with Clq Receptors - Binding to Calreticulin Receptor

Methods

[0227] To demonstrate that PA blocks binding to the calreticulin receptor, the following experiment was performed. Recombinant calreticulin or BSA (negative control) was used to coat a microtiter plate. Clq was then added to the plate alone was incubated with increasing amounts of PA peptide (SEQ ID NO: 3) and then assessed for binding to calreticulin. As a negative control, a 15 amino acid peptide (CP2) that does not bind Clq was used.

[0228] The interaction of labeled Clq with Raji cells was tested by confocal microscopy. Labeled Clq was added to Raji cells. 1.4125 x 10 ⁶ cells/ml were fixed with 4%

paraformaldehyde for 5 min and stored over-night at 4°C. 1 x 10 ⁵ cells stained with: 1 :40 Clq 488 for 20 min, washed x2, and stained with DAPI mounting medium, and stored overnight at 4°C.

[0229] P A-dPEG24 (SEQ ID NO : 21 ) was pre-incubated with C 1 q at two different concentrations before adding to Raji cells. 1.4125 x 10 ⁶ cells/ml were fixed with 4% paraformaldehyde for 5 minutes and stored over-night at 4°C. 1 x 10 ⁵ cells stained with: 1 :40 Clq 488 plus 1.45 mg/ml PA-dPEG24 (SEQ ID NO: 21) (1 :20 of 29 mg/ml) or 2.9 mg/ml PA-dPEG24 (SEQ ID NO: 21) (1 : 10 of 29 mg/ml) for 20 minutes, washed twice, stained with DAPI mounting medium, and stored overnight 4°C.

Results

[0230] PA-dPEG24 (SEQ ID NO: 21) blocked Clq binding to a specific C lq cell receptor (calreticulin/cClqR) in an in vitro assay and also blocked binding of C lq to Raji cells (an immortalized B lymphocyte cell line that is used for evaluating C lq interactions with Clq receptors).

[0231] Clq was added to a plate coated with calreticulin, the results showed that Clq binds specifically to calreticulin but not BSA, as expected (FIG. 15). When plates were coated with Clq, calreticulin and BSA, PA peptide (SEQ ID NO: 3) bound specifically to Clq, the interaction between PA was specific for Clq and not calreticulin or BSA (FIG. 14A). When Clq was incubated with increasing amounts of PA peptide (SEQ ID NO: 3) and then assessed for binding to calreticulin, PA dose-dependently inhibited binding to calreticulin (FIG. 16). As a negative control, a 15 amino acid peptide (CP2) that does not bind Clq did not show competitive inhibition of Clq binding to calreticulin (FIG. 16).

Additionally, PA (SEQ ID NO: 3) inhibited Clq binding to purified calreticulin (FIG. 14B) and Raji cells (FIG 14C-D) consistent with inhibition of Clq binding to surface CRT. The experiment setup is show in FIG. 14E.

[0232] The interaction of labeled Clq with Raji cells was investigated by confocal microscopy. Labeled Clq (FITC) bound to Raji cells (DAPI) as shown in FIGS. 17A-C. When PA-dPEG24 (SEQ ID NO: 21) was preincubated with Clq at two different

concentrations, the amount of labeled Clq bound to Raji cells decreased significantly (FIGS. 18A-C and 19A-C), demonstrating that (SEQ ID NO: 21) inhibited binding of Clq to these cells. Although not bound by any theory, (SEQ ID NO: 21) presumably inhibited binding of Clq by disrupting Clq interaction with a Clq receptor(s) on the cell surface.

Example 15 - Peptide compounds significantly reduced brain injury in a rat model of neonatal hypoxic ischemic encephalopathy

Methods

[0233] P10 Wistar rat pups were subjected to unilateral right carotid ligation (Vannucci model) followed by 8% hypoxia. Experimental animals were injected with 150mg/kg PA- dPEG24 (SEQ ID NO: 21) intraperitoneally. Controls included 1) cobra venom factor (CVF) injection to deplete complement 2) therapeutic hypothermia for 6 hours. Animals were harvested at 4, 8, 16 and 48 hours after intervention. CH50 assay was performed on rat plasma to measure systemic complement activity. Cerebral infarct volumes were measured by staining harvested brain tissue with 2 % TTC using image J software.

Results

[0234] Systemic complement activity was almost completely eliminated in animals injected with CVF for 72 hours after injection as expected. In the PA-dPEG24 (SEQ ID NO: 21) group, systemic complement activation was decreased by 70% at 0.5 hours after injection before gradually returning to baseline at 8 hours (FIG. 30). Hypoxia after carotid ligation yielded 20% infarction. CVF injection before ligation/hypoxia almost completely attenuated brain injury. HT reduces infarction by 50%. Injection with PA-dPEG24 (SEQ ID NO: 21) showed variable neuroprotection (FIG. 31) with an average reduction in infarction by 40%. Although not bound by any theory, SEQ ID NO: 21 may mimic the neuroprotective effect of therapeutic hypothermia by decreasing Clq deposition in the brain up to 48 hours. There was significantly less Clq deposition in the SEQ ID NO: 21 treated group (Normothermia +PIC1 (SEQ ID NO: 21) - triangle) when compared to untreated HIE controls (Normothermia - diamond) at 4, 12, 24 hours after brain injury (FIG. 32A).

[0235] SEQ ID NO: 21 also showed histological evidence of neuroprotection after HIE. Brain histology five days after HIE showed the neuro-preservatory action of SEQ ID NO: 21 similar to therapeutic hypothermia. Neuronal destruction was reduced in those treated with SEQ ID NO: 21, and the treatment also increased viable cells in the brain (FIG. 32B). For instance, in FIG 32B, Panels A-D show cresyl violet staining. Cresyl violet stains Nissl substance in neurons. The HIE group (Panel B) shows a significant decrease in cresyl violet staining compared to the SEQ ID NO: 21 group (Panel D), indicating the neuro-preservatory action of SEQ ID NO: 21 similar to therapeutic hypothermia (Panel C). Panels E-H show hematoxylin and eosin staining. Hematoxylin and Eosin staining demonstrated a greater degree of neuronal destruction (necrosis, pyknosis and karyorrhexis) in the HIE brain (Panels E, F, and G) when compared to those treated with SEQ ID NO: 21 (Panel H). Panels I-L show acridine orange staining. Acridine orange stains viable parts of the brain a bright green. HIE significantly decreases acridine orange staining in the brain (Panel J). SEQ ID NO: 21 treatment restores acridine orange staining, indicating the presence of more viable cells in the brain (Panel L).

[0236] Histological neuroprotection also translated into functional improvement after HIE. In a negative geotaxis test 1-7 days after HIE, animals injected with PICl (SEQ ID NO: 21) performed significantly better (FIG. 32C, green bars) than normothermia HIE animals (FIG. 32C, red bars). The negative geotaxis test is an innate escape response test measuring the time it takes for a pup to climb up when placed head down on a wire mesh.

[0237] This data showed that SEQ ID NO: 21 can reduce brain infarct volumes without prolonged systemic complement depletion, and improved functional outcomes after HIE. Although not bound by any theory, SEQ ID NO: 21 may have decreased Clq deposition in the brain. In some embodiments, SEQ ID NO: 21 is a useful adjunct therapy to HT to improve neurological outcomes in HIE. Example 16 PIC 1 Inhibited Replication of Herpes Simplex Virus Type 1 and Herpes Simplex Virus Type 2

[0238] CV- 1 cells were infected with HS V- 1 -GFP for 16 hours. Post-infection, CV- 1 cells were detected by flow cytometry (FIG. 33). Uninfected cells (negative control, GFP-) result in a left peak, while the peak in infected cells (GFP+) will shift to the right. When PA- PEG24 (SEQ ID NO: 21) was added to the CV-1 cells before infection, it inhibited viral infection in a dose dependent manner. Acyclovir (the standard of care for severe HSV-1 infections) was also added as a control at the same molar ratio. While Acyclovir was able to inhibit HSV-1 replication significantly, it was never able to completely inhibit replication as indicated by the small GFP+ population of cells. This was in contrast to PA-PEG24 (SEQ ID NO: 21), where there was no detection of GFP+ cells at 4-5mM PA-PEG24 (SEQ ID NO: 21). In addition, PA-PEG24 (SEQ ID NO: 21) and Acyclovir showed minimal toxicity in CV-1 cells, which indicated that inhibition of viral replication was not due to cell death (FIG. 33). PA-PEG24 (SEQ ID NO: 21) also inhibited replication of HSV-2 in a dose dependent manner (FIG. 34).

Example 17 - Peptide compounds promoted growth of L. acidophilus and L. leichmannii.

[0239] The ability of PIC 1 to not kill beneficial commensal Lactobacillus (as shown in FIG. 35) greatly enhances the safety of this antibiotic. The most common side effects of current antibiotics are antibiotic-associated diarrhea and yeast infections because the antibiotics kill normal intestinal flora. Among the most dreaded antibiotic side effects is C. difficile colitis, which can be life-threatening and is often recurrent. FIG. 35 shows that PA- PEG24 (SEQ ID NO: 21) can promote growth of Lactobacillus in a dose dependent manner.

Example 18 - PICl had anti-microbial activity.

[0240] Several PICl peptides were anti -bacterial across a broad range of bacterial pathogens. For instance, PA-dPEG24 (also known as AFl, SEQ ID NO: 21) has been shown to inhibit growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae in a dose dependent manner (as shown in FIG. 36A). Additionally,

Pseudomonas aeruginosa growth (FIG. 36B) was suppressed by five variants of PICl (AFl- 5) peptides (SEQ ID NOs: 7, 8, 12, and 21. PICl 's (SEQ ID NO: 21) anti-bacterial activity was further confirmed by confocal microscopy, which showed PICl peptides bound to the outer surface of Staph aureus bacteria (FIG. 36C). [0241] In some embodiments, the peptide compounds inhibited Neisseria gonorrhoeae growth in liquid culture. PA-PEG24 (SEQ ID NO: 21) reduced Neisseria gonorrhoeae growth at 20 mg (FIG. 37 A) or 30 mg (FIG. 37B) of PA-PEG24 (SEQ ID NO: 21) and demonstrated dose-dependent inhibition from 0-30 mg/ml PA-PEG24 (SEQ ID NO: 21) (FIG. 37C). Additionally, colony counts from Neisseria gonorrhoeae incubated with or without PA-PEG24 (SEQ ID NO: 21) showed substantial reduction of Neisseria

gonorrhoeae colonies treated with 30mg/ml of PA-PEG24 (SEQ ID NO: 21).

[0242] In conclusion, the data show that SEQ ID NO: 21, 5, 6, 7 and 8, respectively, had anti -bacterial activity against a broad range of bacterial pathogens.

Example 19 - PICl inhibited myeloperoxidase (MPO) activity of neutrophils.

[0243] Myeloperoxidase (MPO) is an enzyme from neutrophils that creates hypochlorite (bleach) in acute inflammation that damages invading microbes and host cells alike. This enzyme is known to be destructive to host tissues in Cystic Fibrosis (CF) and Hypoxic Ischemic Encephalopathy (HIE). PIC1(SEQ ID NO: 21) was found to inhibit MPO activity in sputum samples (sol) isolated from cystic fibrosis (CF) patients. CF sol samples were incubated in the presence or absence of 20 mg/ml PICl (SEQ ID NO: 21) for 30 minutes followed by addition of TMB for 30 minutes at room temperature (as shown in FIG. 38). MPO activity was then measured by detection of TMB color change in a spectrophotometer at 450 nm. MPO activity was found to be reduced in CF sol samples treated with PICl . Preexisting MPO in the sputum of CF patients could have its enzymatic activity blocked with the peptide compounds (FIGS. 39 in a dose-dependent manner (FIG. 39). Additionally, PA- PEG24 (SEQ ID NO: 21) was able to block MPO enzymatic activity in a lysate of brain or brain membrane preparations of rats subject to hypoxic ischemic encephalopathy (as shown in FIGS. 40-42). MPO activity present in the lysates of purified human neutrophils can also be directly inhibited by PA-PEG24 (SEQ ID NO: 21) (as shown in FIG. 42). FIG. 42 shows that PA-PEG24 (SEQ ID NO: 21) dose-dependently inhibits MPO activity in lysates of purified human neutrophils (PMN). PMN lysates were incubated in the presence of increasing amounts of PA-PEG24 (SEQ ID NO: 21). MPO activity was then measured by detection of TMB color change in a spectrophotometer at 450 nm. Finally titration of the inhibition of purified MPO activity by PA-PEG24 (SEQ ID NO: 21) demonstrated that PA- PEG24 (SEQ ID NO: 21) can directly inhibit MPO (as shown in FIG. 43). These findings demonstrated that PICl has a surprising anti-inflammatory effect that is relevant to both CF and HIE. This shows an alternative mechanism that could explain how SEQ ID NO: 3-47 have anti-inflammatory activity.

Example 20 - Acute Kidney Injury and Rhabdomyolysis: Peptide compounds inhibited hemoglobin production of free radical activity:

[0244] Peptide compounds inhibited the pseudo-peroxidase activity of hemoglobin (Hg) from human red blood cells. Hg, like myeloperoxidase (MPO), contains a heme group that can catalyze peroxide reactions affecting proteins, nucleic acids and lipids that damage host tissues. The pseudo-peroxide activity of Hg is measured using a reaction between hydrogen peroxide (H ₂ 0 ₂ ) and the chromogen tetramethylbenzidine (TMB). The oxidized TMB displays a color change that can be read in a spectrophotometer. Given that PA-PEG24 (SEQ ID NO: 21) could inhibit MPO peroxidase activity, we tested whether the peroxidase activity of Hg from lysed human RBCs could be inhibited by PA-PEG24 (SEQ ID NO: 21) in the same type of experimental assay. To this end, RBC lysates yielding free Hg were incubated with TMB in absence or presence of PA-PEG24 (SEQ ID NO: 21). As shown in FIG. 44, increasing amounts of RBC lysates gave an increase in absorbance demonstrating increased oxidized TMB substrate. In the presence of 20mg/ml PA-PEG24 (SEQ ID NO: 21), there was no detectable oxidized TMB. In FIG. 45, increasing amounts of PA-PEG24 (SEQ ID NO: 21) led to a dose-dependent decrease in oxidized TMB signal. FIG. 46 shows a titration of oxidized TMB with increasing amounts of RBC lysate. In the presence of increasing amounts of PA-PEG24 (SEQ ID NO: 21), oxidized TMB signal decreases dose dependently. Hemolysis leading to the presence of free Hg in human circulation is toxic to the kidneys, resulting in acute kidney toxicity and renal failure. Thus, the data supported that PA-PEG24 (SEQ ID NO: 21) can prevent acute kidney injury by reducing hemolysis and pseudo- peroxidase activity caused by the release of free hemoglobin. Thus, the peptides can prevent acute kidney injury, as well as treat or prevent hemolytic conditions such as, e.g., sickle cell disease, hemolytic transfusion reaction, autoimmune hemolysis, etc.

[0245] To compare the activity of IVIG versus PICl, animals were treated

prophylactically with an equal dose of IVIG or PICl (40 mg/animal) or saline control. Blood of animals that received IVIG or PICl before xenotransfusion were subject to two-color flow cytometry probing for both F human glycophorin A expression and bound C3 fragments. Representative flow cytometry plots of RBCs at 0.5, 5, and 20 minutes post-transfusion are shown for IVIG (FIG. 27A-C) and PICl (FIG. 27D-F) animals. FIG. 28 A shows hemolysis measured as free Hb for animals receiving IVIG or PICl prophylactically compared to saline control. At 30 seconds after transfusion, PICl-treated animals demonstrated increased numbers of C3-negative human RBCs (p 5 0.05) and a 3.6-fold (p 5 0.04) decrease in C3- opsonized human RBCs compared with IVIG treated animals (FIG. 28B). At 30 seconds after transfusion, PICl-treated animals showed a trend toward decreased C3 opsonization of nonhuman (glycophorin A-negative) RBCs (p 5 0.08) compared with IVIG animals (FIG. 28C). Total surviving human RBCs at 20 minutes was increased 1.6-fold (p 5 0.03) for PICl- treated compared with IVIG treated animals (FIG. 28D).

[0246] To ascertain if the intravascular toxic free Hb would cause acute kidney injury, the animals were monitored for 6 hours post-transfusion. In the IVIG prophylaxis group, two of the three animals were noted to exhibit gross hematuria as well as three of four animals in the saline group, but the urine of the PICl-treated animals appeared normal. At necropsy, the kidneys of the IVIG group looked darker and enlarged compared to the kidneys of the PICl- treated group, which appeared normal (FIG. 29B). On measuring the weight of the kidneys individually, the saline group exhibited a significant increase in weight compared to animals receiving PICl (p 5 0.0001) whereas the IVIG group also demonstrated a significant increase in weight compared to animals receiving PICl (p 5 0.005), consistent with edematous kidneys (FIG. 29A). Histopathology sections revealed that the saline and IVIG-treated animals had early acute tubular necrosis, as noted by the disappearing nucleus and nuclear material and tubular edema (saline, FIG. 29C, i-ii; IVIG prophylaxis, FIG. 29C, iii-iv) compared to the slight edema, but otherwise healthy kidney architecture for the PICl animals (PICl prophylaxis, FIG. 29C, v and vi). Taken together, these data demonstrated that PICl inhibits hemolysis of the transfused mismatched RBCs preventing subsequent free Hb- associated acute kidney injury.

[0247] Rhabdomyolysis is a serious syndrome caused by direct or indirect muscle injury resulting from crush injuries, certain infections, use of certain medications, seizures and other events. The resulting injury to tissues causes release of myoglobin, the oxygen-binding protein found in muscle tissue, into the bloodstream. Myoglobin is harmful to kidneys and there is currently no treatment for Rhabdomyolysis. The discovery that PA-PEG24 (SEQ ID NO: 21) can inhibit pseudo-peroxidase activity of hemoglobin (Hg) showed that PA-PEG24 (SEQ ID NO: 21) is efficacious in treating rhabdomyolysis. Because hemoglobin is similar to myoglobin, PA-PEG24's (SEQ ID NO: 21) ability to inhibit hemoglobin suggests its efficacy in inhibiting the peroxidase activity of myoglobin. Example 21 -Toxicity Data for PICl Peptides and Variants

[0248] Single-dose toxicology studies with PA-PEG24 (SEQ ID NO: 21) in rats for both maximum deliverable dose and upper limit of the effective dose range have been performed. For the maximum deliverable dose (limited by maximum achievable concentration of PA- PEG24 (SEQ ID NO: 21) (60 mg [240 mg/kg] i.v.)) and deliverable volume, in the treatment group, multiple blood draws were performed on six animals for 14 days. No toxicities were seen on the blood work (CBC and complete blood chemistry) over the first 24 hours, 48 hours, day 7 or day 14. A small drop in hemoglobin is noted at 48 hours due to multiple blood draws taken during the first 24 hours. Normal histology was found for in the brain, heart, lungs, liver, spleen, kidneys, and pancreas. The most pertinent blood parameters for this experiment are shown in TABLE 1 1.

[0249] Toxicity studies were later performed with larger numbers of animals (n = 18 in the treatment group) using a high-dose at the upper end of the therapeutic range (PA-PEG24 (SEQ ID NO: 21) at 40 mg [160 mg/kg] i.v.). Eight animals were sacrificed at day 3, and ten were sacrificed at day 14. Blood chemistry assay results (CBC, superchem, and coagulation) showed no differences between pretreatment, day 3 or day 14 values (TABLE 12). There was no histological evidence for toxicity in any organ (including brain, heart, lungs, liver, spleen, kidneys, and pancreas) at either time point. Thus, single-dose toxicological evaluation of SEQ ID NO: 21 revealed no safety concerns.