CN116528883A - Peptides and methods of use thereof - Google Patents

Abstract

The present invention provides synthetically modified peptides as Polar Assortant (PA) peptides including C-terminal PEGylation. The invention also provides methods of using at least one synthetic peptide to modulate the complement system and interact with neutrophils to alter their binding and activity.

Description

Peptides and methods of use thereof

Cross-reference to related applications

The present application claims priority from U.S. provisional application Ser. Nos. 63/085,556 and 63/185,831 filed on 7.5/2021, 30/9/2020, the disclosures of which are incorporated herein by reference in their entireties.

Sequence listing

The present application contains a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy generated at month 9 of 2021 is named 251110_000139_sl.txt and is 1,228 bytes in size.

Technical Field

Embodiments of the present invention relate generally to synthetic peptides and their use for therapy and diagnosis, and more particularly to pegylated forms of the synthetic peptides.

Background

Complement system

The complement system is an essential component of the innate immune system, plays a key role as a defense mechanism against invading pathogens, elicits adaptive immune responses, and helps clear immune complexes and apoptotic cells. Three different pathways constitute the complement system: classical pathway, lectin pathway and alternative pathway. C1q and mannose-binding lectin (MBL) are structurally related recognition molecules of the classical and lectin pathways, respectively. IgM or aggregated IgG acts as the primary ligand for C1q, while MBL recognizes polysaccharides such as mannans. Ligand binding of C1q and MBL results in sequential activation of C4 and C2, forming classical and lectin pathway C3 convertases, respectively. In contrast, alternative pathway activation does not require recognition molecules, but can amplify C3 activation initiated by the classical or lectin pathway. Activation of any of these three pathways results in the formation of inflammatory mediators (C3 a and C5 a) and Membrane Attack Complexes (MACs), leading to cell lysis.

Although the complement system plays a key role in many protective immune functions, complement activation is an important mediator of tissue damage in a wide range of autoimmune and inflammatory disease processes. (Ricklin and Lambris, "Complement-targeted therapy" (complete-targeted therapeutics), nat Biotechnol 2007;25 (11): 1265-75).

There is a need for complement modulators. On the one hand, the complement system is an important host defense against pathogenic organisms. On the other hand, its unchecked activation can lead to devastating host cell damage. Currently, although complement dysregulation is known to be associated with morbidity and mortality in many disease processes including autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, and multiple sclerosis, only two anticomplementary therapies have recently been approved for use in humans: 1) Ekulizumab (Soliris) ^TM ) And 2) ultomiris (Ravulizumab) ^TM ) These are two humanized long-acting monoclonal antibodies directed against C5 for the treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). PNH and aHUS are orphan diseases with very few patients. Currently, no complement regulator is approved for the more common disease processes in which complement activation disorders play a critical role. Complement activation disorders can play a role in chronic disease indications and acute disease indications.

There is a need to develop peptides that inhibit the classical, lectin and alternative pathways of the complement system, as each of these three pathways has been shown to have an impact on a number of autoimmune and inflammatory disease processes. Specific blockade of the classical and lectin pathways is particularly desirable because both pathways are associated with ischemia reperfusion-induced injury and other diseases in many animal models. Humans with alternative pathway defects suffer from severe bacterial infections. Thus, a functional alternative approach is essential for immune surveillance against invading pathogens.

The PIC1 family of molecules comprises a series of rationally designed peptides based on the outer coat protein of the random astrovirus that possess several anti-inflammatory functional properties, including inhibition of the complement classical pathway, myeloperoxidase inhibition, neutrophil Extracellular Trap (NET) inhibition, and antioxidant activity. The original compound is a 15 amino acid peptide sequence IALILEPICCQERAA (SEQ ID NO: 1) with a C-terminal monodisperse 24-mer PEGylation moiety (IALILEPICCQERAA-dPEG 24; PA-dPEG24; SEQ ID NO: 2) to increase its water solubility. Other features of the PA-dPEG24 molecule are discussed herein.

Complement system and ocular diseases

The complement system has a positive role in maintaining immune homeostasis and protecting the eye from pathogens, which involves complex interactions between complement activating and complement modulating molecules to control potential infections. Although the complement system is essential for immune supervision, excessive and deregulated complement activation is associated with a number of inflammatory and keratitis diseases in the eye, such as autoimmune and infectious uveitis, acute Macular Degeneration (AMD), dry Eye (DED), infectious and non-infectious keratitis, corneal damage and repair, retinopathy of prematurity (ROP), ocular graft versus host disease (GvHD), diabetic retinopathy (Jha et al, 2007), macular edema after Retinal Vein Occlusion (RVO) and Diabetic Macular Edema (DME).Neutral Granulocytes, neutrophil extracellular trap net and eye diseases

It has recently been reported that neutrophils have been shown to play a key role in the pathogenesis of AMD, as seen in mouse models and ex vivo studies of the human eye of cadavers of AMD patients (Ghosh et al, 2019). In these studies, elevated interferon lambda was identified in both human and mouse eyes, and this high expression of interferon lambda induced migration of neutrophils from the venous circulation to the retina, ultimately leading to pathological damage of the eye.

In addition to the general role of neutrophils in ocular diseases, neutrophil Extracellular Traps (NET) have been shown to play a pathogenic role specifically in a variety of other ocular diseases such as chronic inflammation of the cornea, DED, infectious keratitis, corneal damage, ocular GvHD, non-infectious uveitis (e.g., behcet's disease), and infectious uveitis, diabetic retinopathy, and finally AMD (Estua-Acosta et al, 2019; ghosh et al, 2019). In particular, NETosis biomarkers, myeloperoxidase (MPO), neutrophil elastase, and citrullinated histone H3 have been shown to have an effect on pathogenesis in mouse models of AMD in the case of AMD (Ghosh et al, 2019).

Complement system and Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS)

ALI is often a severely traumatic complication that can develop into ARDS, leading to significant morbidity and mortality [ M ca J, jor O, holub M, sklienka P,f, burda M et al, "ARDS mortality in the past and present: system review "(Past and Present ARDS Mortality Rates: A Systematic Review), respir Care 2017;62 (1):113-122]. To date, there is no pharmacological intervention to prevent ALI, and current standards of care are supportive in nature. ALI may be produced by the patient' S underlying clinical condition (e.g., inflammation, trauma, hypotension) and secondary attacks such as Transfusion (Transfusion-associated ALI (trani), resuscitation, radiation) [ Cho MS, modi P, sharma S, "Transfusion-associated acute lung injury" (transfusions-related Acute Lung Injury), 2020, in StatPearls, trends Island (FL): statPearls Publishing;2020 Jan; kumar AK, anjum F, "Ventilator-induced lung injury (VILI)" (venturi-Induced Lung Injury (VILI)), 2020 Dec 15 in StatPearls, treasure Island (FL): statPearls Publishing;2020 Jan; arroyo-Hernandez M, maldonado F, lozano-Ruiz F, ++ >W,/>M, arieta O, "radiation induced lung injury: current evidence "(Radiation-induced lung injury: current evaluation), BMC Pulm Med2021;21 (1):9]Or viral pneumonia (e.g., influenza, respiratory syncytial virus, or coronavirus-related ALI) [ klamp M, ghosh S, mohammed S, nadem Khan M, "how influenza promotes lung injury from virus to inflammation" (From virus to inflammation, how influenza promotes lung damage), J Leukoc Biol 2020; sep 8; alvarez AE, marson FA, bertuzzo CS, arns CW, ribeiro JD, "associated with severity of acute viral bronchiolitis caused by respiratory syncytial VirusEpidemiological and genetic characterization "(Epidemiological and genetic characteristics associated with the severity of acute viral bronchiolitis by respiratory syncytial virus), J petiatr (Rio J) 2013;89 531-43; lee C, choi WJ, "outline the pathogenesis of COVID-19 inflammation from a therapeutic perspective" (Overview of COVID-19 inflammatory pathogenesis from the therapeutic perspective), arch Pharm Res 2021; jan 4:1-18]Is caused by the combination of (a) and (b). Although secondary attacks may vary, the rapidly progressive disease process leading to lung failure is often mediated by exaggerated and overwhelming innate immunity or inflammatory responses driven by excessive complement and neutrophil mediated inflammatory responses. In addition to ALI, dysregulation of neutrophil and complement activation is a key mediator of acute exacerbation in chronic lung diseases such as COPD and steroid resistant neutrophil asthma [ Pandya PH, wilkes DS, "complement system in lung disease" (Complement system in lung disease), am J Respir Cell Mol Biol 2014;51:467-473; khan MA, nicols MR, surguladze B, saadon I, "complement component as potential therapeutic target for asthma treatment" (Complement components as potential therapeutic targets for asthma treatment), respir Med 2014;108:543-549 ]。

In the case of trani, which represents one of the major causes of transfusion-related death, this disease process is complex and not fully understood, but it is currently believed that the "two-shot" model most accurately accounts for the clinical situation, the first shot being mediated by the underlying clinical condition of the patient, the second shot being triggered by a component in the transfusion unit [ sillliman CC, paterson AJ, dickey WO, strock DF, popovsky MA, caldwell SA et al, "association of bioactive lipids with the occurrence of transfusion-related acute lung lesions: retrospective studies "(The association of biologically active lipids with the development of Transfusion-related acute lung injury: a retrospective study), transfusions 1997;37 719-26; sillliman CC, mcLaughlin NJ, "Transfusion-associated acute lung injury" (transfusions-related acute lung injury), blood Rev 2006;20 (3):139-59]. Various in vitro, in vivo and ex vivo studies have shown that neutrophils cause Acute Lung Injury (ALI) through direct activation, formation of Reactive Oxygen Species (ROS) and formation of Neutrophil Extracellular Trap (NET), play a key role in the pathogenesis of trani [ Rebetz J, semole JW, kapur R, "pathogenic role of neutrophils in acute respiratory distress syndrome and transfusion-related acute lung injury" (The Pathogenic Involvement of Neutrophils in Acute Respiratory Distress Syndrome and Transfusion-Related Acute Lung Injury), transfus Med Hemother2018;45 (5):290-298]. Furthermore, it has been previously hypothesized that the complement system may lead to neutrophil activation and ROS and NET formation through C3a and C5a interactions with neutrophils, thereby playing a role in trail [ Jongerius I, porcelijn L, van Beek AE, sample JW, van der Schoot CE, vlaar APJ, et al, "role of complement in transfusion-related acute lung injury" (The Role of Complement in Transfusion-Related Acute Lung Injury), transfus Med Rev 2019;33 (4):236-242].

Asthma (asthma)

Bronchial asthma is a chronic, heterogeneous, inflammatory disease mediated by different immunopathogenic mechanisms, including eosinophilic, neutrophilic, mixed granulocyteic and oligogranulocyteic asthma. It is estimated that between 3.6-10% of asthmatics suffer from severe, uncontrolled diseases refractory to corticosteroids and β2 agonists, which represent standard drugs for the treatment of asthma [ Syabbalo N (2020), "clinical characterization and management of neutrophilic asthma" (Clinical Features and Management of Neutrophilic Asthma), J Pulm Med Respir Res 6:036]. Neutrophilic asthma is the most common form of acute severe asthma seen in adults. Neutrophilic asthmatics are characterized by frequent emergency department visits, hospitalizations, and tracheal intubation in about 23% of patients due to sudden fatal asthma [ Fahy JV, kim KW, liu J, boushey HA (1995), "significant neutrophilic inflammation in sputum from asthmatic exacerbating subjects" (Prominent neutrophil inflammation in sputum from subjects with asthma exacerbations), J Allergy Clin Immunol 95:843-852; sur S, crotty TB, kephart GM, hyama BA, colby TV et al (1993), "bursty fatal asthma: is a unique entity with fewer eosinophils and relatively more neutrophils in the airway mucosa? "(Sudden-onset fatal asthma: A distinct entity with few eosinophils and relatively more neutrophils in the airway mucosa. Currently, the inability to control steroid refractory neutrophilic asthma represents an unmet clinical need.

The pathophysiological role of neutrophils in severe asthma has been demonstrated in human ex vivo studies. Recently, NET, extracellular DNA and other neutrophil derived products have been considered as possible biomarkers and therapeutic targets for severe asthma [ Lachowicz-Scroggins ME, dunican EM, charbit AR, raymond W, looney MR, peters MC et al (2019), "extracellular DNA in severe asthma, neutrophil extracellular trap and inflammatory body activation" (Extracellular DNA, neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma), am J Respir Crit Care Med.199 (9): 1076-1085; varrichi G, modestino L, poto R, cristinizino L, gentile L, postingione L et al (2021), "neutrophil extracellular trap and neutrophil derived mediators as possible biomarkers in bronchial asthma" (Neutrophil extracellular traps and neutrophil-derived mediators as possible biomarkers in bronchial asthma), clin Exp Med.2021Aug 3.Doi:10.1007/s10238-021-00750-8].

The inventors found that PIC1 peptide can modulate neutrophil activity and thus evaluated the efficacy of RLS-0071 in a rat model of neutrophilic asthma using Ovalbumin (OVA) and Lipopolysaccharide (LPS) allergens.

Angiogenesis

Angiogenesis is the process of forming new blood vessels from pre-existing blood vessels and continues the growth of the vascular system through the processes of sprouting and division. Angiogenesis is a normal physiological process in growth and development and plays a key role in wound healing and granulation tissue formation. However, it is also a key factor in tumor growth and plays a pathogenic role in many ocular diseases such as Acute Macular Degeneration (AMD), retinopathy of prematurity (ROP) and diabetic retinopathy, resulting in the use of angiogenesis inhibitors in the treatment of cancer and ophthalmic diseases, respectively. VEGF is a major participant in the process of angiogenesis, and many angiogenesis inhibiting drugs target VEGF. However, VEGF-independent angiogenesis also occurs in a variety of different inflammatory disease states. Thus, the inventors hoped to investigate the effect of PIC1 peptide on VEGF.

There is a need in the art for peptide-based inhibitors of different pathways of the complement system. There is also a need in the art for therapeutic peptides that treat ophthalmic diseases and/or conditions, ALI and/or ARDS, asthma and modulate angiogenesis.

Disclosure of Invention

As described in the background section, there is a great need in the art for techniques to identify peptide-based inhibitors for different pathways of the complement system and to exploit this understanding to develop new therapeutic peptides. The present invention meets this and other needs. Embodiments of the present invention relate generally to synthetic peptides, and more particularly to pegylated synthetic peptides and their use in methods of modulating the complement system and interacting with neutrophils to modulate their binding and other activities.

In one aspect, the invention provides synthetic peptides that modulate the complement system and methods of using these peptides. In particular, in certain embodiments, the synthetic peptides can bind, modulate, and inactivate C1 and MBL, and thus can efficiently inhibit activation of the classical and lectin pathways at the earliest point in the complement cascade, while leaving the alternative pathway intact. These peptides are of therapeutic value because they selectively modulate and inhibit C1 and MBL activation without affecting alternative pathways, and are useful in the treatment of diseases mediated by deregulated classical and lectin pathway activation. In other embodiments, the peptide modulates classical pathway activation, but does not modulate lectin pathway activation. The peptides may be used in a variety of different therapeutic indications.

In other embodiments, the synthetic peptide is capable of altering cytokine expression, including but not limited to cytokine expression in ALI and/or ARDS models.

In other embodiments, the synthetic peptide is capable of inhibiting or altering neutrophil binding and/or adhesion.

In other embodiments, the synthetic peptide is capable of increasing neutrophil survival.

In other embodiments, the synthetic peptide may bind to a cell surface receptor in vivo, such as, but not limited to, integrins and intercellular adhesion molecules (ICAM).

In certain embodiments, the invention is based on the identification and modification of 15 amino acid peptides derived from Polar Assortant (PA) peptide (SEQ ID NO: 1), derivatives of the peptides and methods of their use. The PA peptide is an disordered peptide derived from a human astroviral protein known as CP 1. PA peptide is also known as PIC1 (peptide inhibitor of complement C1), astroFend, AF or SEQ ID NO:1. the PIC1 peptide was originally so named because it was found to be associated with diseases mediated by the complement system. The PEGylated form of the PIC1 peptide, designated PA-dPEG24 (SEQ ID NO:2; RLS-0071), has 24 PEG units on the C-terminus of the peptide and exhibits improved solubility in aqueous solutions. A sarcosine substitution form of the PIC1 peptide, designated PA-I8Sar (SEQ ID NO:3; RLS-0088), has a sarcosine at position 8 of the peptide to replace isoleucine.

In one aspect, the invention provides a method of altering cytokine expression, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In one aspect, the invention provides a method of inhibiting or altering neutrophil binding and/or adhesion, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In one aspect, the invention provides a method of increasing neutrophil survival, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In one aspect, the invention provides a method of inhibiting or altering the binding of neutrophils to a cell surface receptor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In one aspect, the invention provides a method of treating a disease or disorder characterized by altered expression of a cell surface receptor, the method comprising administering a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In one aspect, the invention provides a method of treating and/or preventing ALI and ARDS comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In one aspect, the invention provides a method of treating and/or preventing an ocular disease and/or disorder characterized by a disorder of complement activation and/or neutrophil regulation, the method comprising administering a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2, and a synthetic peptide. In certain embodiments, the ocular disease or disorder is characterized by complement inhibition and/or inhibition of myeloperoxidase activity or NETosis. In certain embodiments, the ocular disease or disorder is autoimmune and infectious uveitis, acute Macular Degeneration (AMD), dry Eye (DED), infectious and non-infectious keratitis, corneal damage and repair, retinopathy of prematurity (ROP), ocular graft versus host disease (GvHD), diabetic retinopathy, macular edema after Retinal Vein Occlusion (RVO), and Diabetic Macular Edema (DME).

In one aspect, the invention provides a method of treating asthma, the method comprising administering a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2, and a synthetic peptide. In certain embodiments, the asthma is severe asthma, steroid refractory asthma or neutrophilic asthma.

In one aspect, the invention provides a method of modulating angiogenesis, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In an embodiment of any of the above methods, the composition further comprises at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient. In an embodiment of any of the above methods, the SEQ ID NO:2 and/or 3 is from about 10mg/kg to about 160mg/kg. In an embodiment of any of the above methods, the SEQ ID NO:2 and/or 3 is from about 20mg/kg to about 160mg/kg. In an embodiment of any of the above methods, the SEQ ID NO:2 and/or 3 is about 40mg/kg to about 160mg/kg. In an embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO:2 and/or 3 in at least one dose, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3. In an embodiment of any of the above methods, the administering comprises a therapeutically effective amount of SEQ ID NO:2 and/or 3 comprising from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3. In an embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO:2 and/or 3 in two doses, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3, and the second dose comprises from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3. In certain embodiments, the second dose is administered 30 seconds to 3 hours after the administration of the first dose. In an embodiment of any of the above methods, the composition is formulated for ocular administration. In one embodiment, the composition further comprises an ophthalmically acceptable carrier and/or excipient. In an embodiment of any of the above methods, the ocular administration comprises topical administration, periocular injection, subconjunctival injection, aqueous humor injection, intraocular injection, intravitreal injection, or introduction of an intracorneal or intraocular implant. In an embodiment of any of the above methods, the composition is formulated for nasal administration. In an embodiment of any of the above methods, the nasal administration comprises inhalation, insufflation, or nebulization. In an embodiment of any of the above methods, the nasal composition is in the form of a spray, solution, gel, cream, lotion, aerosol, or nebulizer solution, or as an insufflation superfine powder.

In an embodiment of any of the above methods, the cell surface receptor comprises an integrin or an intercellular adhesion molecule (ICAM). In an embodiment of any of the above methods, the ICAM comprises ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5. In an embodiment of any of the above methods, the disease or disorder is characterized by an increase in at least one of ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5.

These and other objects, features and advantages of the present invention will become more fully apparent from the following description, appended claims and accompanying drawings.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

FIG. 1 shows that Intravenous (IV) administration of PA-dPEG24 (also referred to herein as RLS-0071), delivered either before or after incompatible erythrocyte infusion, reduced the levels of IFNγ, IL-6, IL-2, IL-10, TNF α, MCP-1, RANTES, MIP1 α, IL-1 β, MIP-2. Cytokine levels obtained from sham operated animals and animals receiving only LPS, lps+30% infusion and single doses of 10 or 160mg/kg RLS-0071 administered prior to infusion (prophylactic) or single doses of 40 and 160mg/kg RLS-0071 administered post infusion (rescue) were determined by xMAP bead-based immunoassays. Data are mean and standard error of mean. * P <0.05 is indicated, P <0.01 is indicated, compared to lps+30% infusion.

FIG. 2 shows that Intravenous (IV) administration of RLS-0071 delivered either before or after incompatible erythrocyte infusion reduced the levels of IL-5, IL-18, IL-1α, IL-13, IL-17, IL-12 and IP-10. Cytokine levels obtained from sham operated animals and animals receiving only LPS, lps+30% infusion and single doses of 10 or 160mg/kg RLS-0071 administered prior to infusion (prophylactic) or single doses of 40 and 160mg/kg RLS-0071 administered post infusion (rescue) were determined by xMAP bead-based immunoassays. Data are mean and standard error of mean. * P <0.05 is indicated, P <0.01 is indicated, compared to lps+30% infusion.

Figure 3 shows that Intravenous (IV) administration of RLS-0071 delivered either before or after incompatible erythrocyte infusion did not significantly affect the level of anti-inflammatory cytokine IL-4. IL-4 obtained from sham operated animals and animals receiving only LPS, LPS+30% infusion and single doses of 10 or 160mg/kg RLS-0071 administered prior to infusion (prophylactic) or single doses of 40 and 160mg/kg RLS-0071 administered post infusion (rescue) were assayed by an xMAP bead-based immunoassay. Data are mean and standard error of mean.

FIG. 4 shows the effect of Intravenous (IV) administration of RLS-0071 on EGF, LIX, VEGF, leptin, GRO, fractal chemokines, GM-CSF, eosinophil chemokines, and G-CSF levels delivered either before or after incompatible erythrocyte infusion. Cytokine and growth factor levels obtained from sham operated animals and animals receiving only LPS, lps+30% infusion and single doses of 10 or 160mg/kg RLS-0071 administered prior to infusion (prophylactic) or single doses of 40 and 160mg/kg RLS-0071 administered post infusion (rescue) were determined by xMAP bead-based immunoassays. Data are mean and standard error of mean. * P <0.05 is indicated, P <0.01 is indicated, compared to lps+30% infusion.

FIGS. 5A-B show RLS-0071 staining of liver (5A) and kidney (5B) tissue in rats receiving 400mg/kg of PA-dPEG24 administered Intravenously (IV) compared to untreated animals. Liver (5A) and kidney (5B) tissue sections were RLS-0071 stained and visualized by microscopy at 20X and 40X magnification. Brown staining indicates the presence of RLS-0071 in the tissue. Red arrows in liver sections indicate punctate RLS-0071 staining.

FIG. 6 shows immunofluorescent staining of RLS-0071, confirming binding of the peptide to human neutrophils. Human neutrophils were adhered to the slide, fixed with paraformaldehyde, and then incubated in the presence or absence of RLS-0071. Slides were then stained with antibodies against RLS-0071 (chicken anti-PIC 1 antibody), followed by a labeled secondary antibody (anti-chicken antibody, alexa Fluor 488) and counterstained with DAPI. The cells were then observed by microscopy.

FIG. 7 shows that RLS-0071 inhibits adhesion of human neutrophils to the slide. The images show human neutrophils adhering to the slide surface in the presence of increasing concentrations of RLS-0071. Neutrophils were stained with DAPI and imaged with a fluorescence microscope. Representative images are shown. The graph in the lower right panel shows the number of neutrophils adhering to the slide after incubation with increasing concentrations of RLS-0071, followed by washing with PBS, then placing on the slide and incubating for 2.5 hours. His means cells treated with histidine buffer alone (pH 6.5). Data are mean ± SEM of n=4.

FIG. 8 shows that RLS-0071 inhibits adhesion of human neutrophils to slides treated with and without fibrinogen. The images show human neutrophils adhering to the surface of fibrinogen-coated slides in the presence of increasing concentrations of PA-DPEG 24. Neutrophils were stained with DAPI and imaged with a fluorescence microscope. Representative images are shown. The graph in the lower right panel shows the number of neutrophils after incubation with increasing concentrations of RLS-0071, followed by washing with PBS, then placing on fibrinogen coated or untreated slides and incubation for 2.5 hours.

FIG. 9 shows that RLS-0071 increases human neutrophil survival as measured by the CCK8 assay, which measures cell respiration as an indicator of survival (number of living cells). RLS-0071 dose-dependently increased human neutrophil survival in CCK8 assay. Cells in PBS or RPMI were incubated with increasing amounts of RLS-0071. "fresh" means that the cells were not manipulated, plated immediately after the purification process was completed and incubated with CCK8 for 2 hours at 37 ℃.

FIG. 10 shows that RLS-0071 can bind to both the neutrophil receptor LFA-1 and the epithelial cell receptor ICAM-1. RLS-0071 selectively binds to purified endothelial and neutrophil receptors. The plates were coated with purified neutrophil receptors LFA-1 and MAC-1 and endothelial cell receptors ICAM-1 and ICAM-2 and then incubated in buffer with increasing amounts of RLS-0071. The plates were washed, then incubated with rabbit anti-RLS 0071 antisera, washed, then incubated with anti-rabbit antibody HRP. The plate was again washed and developed. Absorbance was read at 450 nm. Pic1=rls-0071. C1q was used as a positive control for RLS-0071 binding.

FIG. 11 shows that RLS-0071 can bind to epithelial cell receptors ICAM-1, ICAM-3, ICAM-4 and ICAM-5. Plates were coated with purified neutrophil receptors ICAM-1, ICAM-2, ICAM-3, ICAM-4 and ICAM-5 and then incubated in buffer with increasing amounts of RLS-0071. The plates were washed, then incubated with rabbit anti-RLS 0071 antisera, washed, then incubated with anti-rabbit antibody HRP. The plate was again washed and developed. Absorbance was read at 450 nm. C1q was used as a positive control for RLS-0071 binding and ICAM-2 was used as a negative control.

FIG. 12 shows that RLS-0071 can bind to the neutrophil receptor LFA-1 or endothelial cell receptor ICAM-1 in plasma. Plates were coated with purified receptor and then incubated with increasing amounts of RLS-0071 in human plasma. The plates were washed, then incubated with affinity purified rabbit anti-RLS 0071 antisera, washed, and then incubated with anti-rabbit antibody HRP. The plate was again washed and developed. Absorbance was read at 450 nm. Pic1=rls-0071. C1q and MPO (myeloperoxidase) were used as positive controls for RLS-0071 binding.

FIG. 13 shows the pooled plasma radiochromatograms at time points from male Sprague-Dawley rats following a single IV administration of 20mg/kg of [14C ] -PIC 1-RLS-0071.

FIG. 14 shows the pooled plasma radiochromatograms at time points from male Sprague-Dawley rats following a single IV administration of 200mg/kg of [14C ] -PIC 1-RLS-0071.

FIG. 15 shows that RLS-0071 does not interfere with the binding of the C1 q-immune complex to receptors on human monocytes. Human monocytes were purified and allowed to attach to microtiter plates. The heat-aggregated human immune complex was allowed to bind to C1q in the presence of increasing amounts of RLS-0071. These complexes were then allowed to bind to monocytes, then washed, and the bound C1 q/immune complexes were detected by primary antibody, then secondary antibody-HRP, and developed with TMB. Absorbance was read at 450 nm. N=3. Bars represent Standard Error of Mean (SEM).

FIGS. 16A-16C show that RLS-0071 reduces the level of inflammatory cytokines in blood. The levels of cytokines IL-1a, IFN-g, IL-1B, IL-6 (16A), IL-17, IL-18, TNFa and RANTES (16B), IL-4, IL-10 and VEGF (16C) from the terminal blood draws of the following experimental groups were determined by an xMAP bead-based immunoassay: false operation, only first hit, 2 hits, 2 hits+10 mg/kg prophylactic dose of RLS-0071,2 hits+160 mg/kg prophylactic dose of RLS-0071, and 2 hits+40 mg/kg rescue dose of RLS-0071 and 2 hits+160 mg/kg rescue dose of RLS-0071. For clarity, only the data for the salvaged drug administration are shown. Data are mean and standard error of mean. * P <0.05 is indicated compared to animals receiving 2 challenge shots.

FIGS. 17A-17C show that RLS-0071 reduces the level of inflammatory chemokines in blood. The levels of the chemokines (17A) MCP-1, (17B) MIP-1a and (17C) MIP-2 from the terminal blood draws from the following experimental groups were determined by an xMAP bead-based immunoassay: false operation, 1 st hit, 2 hits, 2 hits+10 mg/kg of prophylactic dose of RLS-0071,2 hits+160 mg/kg of prophylactic dose of RLS-0071, and 2 hits+40 mg/kg of rescue dose of RLS-0071 and 2 hits+160 mg/kg of rescue dose of RLS-0071. For clarity, only the data for the salvaged drug administration are shown. Data are mean and standard error of mean. * P <0.05 is indicated compared to animals receiving 2 challenge shots.

Fig. 18A-18K illustrate that prophylactic or rescue administration of RLS-0071 reduces acute lung injury. Representative histology of rat lung (H & E staining). (18A) sham surgical controls, (18B) only first hit. (18C) 2 hits, (18D) 2 hits+10 mg/kg of prophylactic dose of RLS-0071, (18E) 2 hits+40 mg/kg of prophylactic dose of RLS-0071, (18F) 2 hits+160 mg/kg of prophylactic dose of RLS-0071, (18G) 2 hits+40 mg/kg of rescue dose of RLS-0071 at 0.5min, (18H) 2 hits+40 mg/kg of rescue dose of RLS-0071 at 60min, (18I) 2 hits+40 mg/kg of rescue dose of RLS-0071 at 90min, (18J) 2 hits+40 mg/kg of rescue dose of RLS-0071 at 120min, and (18K) 2 hits+40 mg/kg of rescue dose of RLS-0071 at 180 min. Bars represent 100 μm. Tissues were observed using a microscope (BX 50, olympus) at room temperature at 20X magnification. Images were acquired using a digital camera (DP 70, olympus).

FIG. 19 shows that prophylactic or rescue administration of RLS-0071 reduces neutrophil mediated lung injury. The H & E stained lung tissue images were turned black and white and quantified by ImageJ analysis. The ratio of black to white pixels is calculated and used as a measure of lung injury (Y-axis). Sham control animals (n=3), only the first hit (n=2), 2 hits (n=3), 2 hits+10 mg/kg of prophylactic dose RLS-0071 (n=4), 2 hits+40 mg/kg of prophylactic dose RLS-0071 (n=6), 2 hits+160 mg/kg of prophylactic dose RLS-0071 (n=9), 40mg/kg of rescue dose RLS-0071 (n=4) at 2 hits+0.5 min, 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+60 min, 40mg/kg of rescue dose RLS-0071 (n=5) at 2 hits+90 min, 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+120 min, and 40mg/kg of rescue dose RLS-0071 (n=3) at 2 times+180 min. For each animal, 10 or more images per slide were quantified. Data are mean and standard error of mean. Statistical analysis was performed using a generalized linear model. * P=0.002 and p <0.001 is indicated compared to 2 animals hit.

FIG. 20 shows that RLS-0071 inhibits complement activation. Plasma was isolated from sham operated animals (n=3) and the following components before the first hit (0 min) and at 5min and 1 hr: only the first hit (n=3), 2 hits (n=3), 2 hits+10 mg/kg of prophylactic dose RLS-0071 (n=8), 2 hits+40 mg/kg of prophylactic dose RLS-0071 (n=4), 2 hits+160 mg/kg of prophylactic dose RLS-0071 (n=5), 40mg/kg of rescue dose RLS-0071 (n=5) at 2 hits+0.5 min, 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+60 min, 40mg/kg of rescue dose RLS-0071 (n=5) at 2 hits+90 min, 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+120 min, and 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+180 min. The C5a in each sample was then measured by ELISA and absorbance was read at 450 nm. Two replicates were measured for each animal at each time point. Data are mean and standard error of mean. Statistical analysis was performed using bootstrap or Welch's ANOVA. * P=0.010, p=0.004, p=0.002, p.ltoreq.0.001, compared to 2 animals challenged.

FIG. 21 shows that RLS-0071 reduces free DNA levels in blood. Plasma was isolated from sham operated animals (n=3) and the following components 4 hours after the start of the experiment: only the first hit (n=3), 2 hits (n=3), 2 hits+10 mg/kg of prophylactic dose RLS-0071 (n=9), 2 hits+40 mg/kg of prophylactic dose RLS-0071 (n=4), 2 hits+160 mg/kg of prophylactic dose RLS-0071 (n=5), 40mg/kg of rescue dose RLS-0071 (n=4) at 2 hits+0.5 min, 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+60 min, 40mg/kg of rescue dose RLS-0071 (n=5) at 2 hits+90 min, 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+120 min, and 40mg/kg of rescue dose RLS-0071 (n=3) at 2 hits+180 min. Plasma samples were incubated with PicoGreen. Fluorescence was read in a microplate reader at 485nm excitation wavelength and 520nm emission wavelength. All free DNA measurements for each animal were performed in triplicate. Data are mean and standard error of mean. Statistical analysis was performed using bootstrap or wilgi variance analysis. * P=0.026, p=0.039, p=0.005 compared to 2 animals hit.

Figures 22A-22D show that RLS-0071 delivered by Intravitreal (IVT) injection has a longer half-life than RLS-0071 administered Intravenously (IV). (22A) and (22B): rats dosed with 160mg/ml RLS-0071IVT were euthanized at the indicated time points and the vitreous humor isolated. (22C) and (22D): rats dosed with 200mg/ml RLS-0071IV were bled and plasma isolated at the indicated time points. The vitreous and plasma samples were then analyzed in a sandwich ELISA to detect the level of RLS-0071. Fig. 22B and 22D correspond to fig. 22A and 22C, respectively, and the Y-axis is exaggerated to emphasize peptide levels at later time points.

FIG. 23 shows that RLS-0071 delivered by IVT stained retinal tissue 1 hour after administration. Rats were injected with saline or 160mg/kg RLS-0071 IVT. Animals were euthanized 5 minutes after saline infusion or 1 hour after RLS-0071 infusion, and eyes were histologically treated and stained with antibodies to RLS-0071, and then detected by DAB staining. Images were observed by microscopy at 4X (upper panel) and 20X (lower panel) magnification 5 minutes after IVT (left panel) and 1 hour after IVT (right panel).

Figure 24 shows the C5a production measured in plasma of a two-hit rat model of acute lung injury.

Figure 25 shows that incompatible erythrocytes infused as the second hit in the 2 hit ALI model activate the classical complement pathway, causing hemolysis, releasing free hemoglobin into the blood, which can be measured in the plasma. Animals treated with saline are shown in the middle column and RLS-0071 animals are shown in the right column. On the left column are non-infused sham operated animals.

Fig. 26 shows the results of CH50 measurement of plasma obtained from 2 ALI animals. Saline-treated animals are shown in the right column and RLS-0071 animals are shown in the left column.

Fig. 27 shows the experimental design for testing the effect of RLS-0071 on severe asthma.

FIG. 28 shows that RLS-0071 reduces neutrophil levels in bronchoalveolar lavage fluid (BALF) in asthmatic rats. Upper graph: representative BALF images for each experimental group are shown: sham control, animals receiving intraperitoneal Ovalbumin (OVA)/Lipopolysaccharide (LPS) regimen (asthma, day 24), asthmatic animals receiving prophylactic doses of 160mg/kg RLS-0071 on days 21, 22 and 23, and animals receiving rescue doses of 160mg/kg RLS-0071 on days 22 and 23. All animals were sacrificed and BALF was collected on day 24. BALF was observed by microscopy (BX 50, olympus) at room temperature under 40X magnification. Images were acquired using a digital camera (DP 70, olympus). The following figures: white blood cells were quantified by two independent observers. Cell counts are expressed as a percentage of the total. Data are mean and standard error of mean. * P <0.03 is indicated compared to asthmatic animals.

FIG. 29 shows that RLS-0071 reduces protein levels in BALF in asthmatic rats. The following experimental groups were evaluated: sham animals (unstimulated), animals receiving OVA/LPS regimen (asthma), asthmatic animals receiving prophylactic doses of 160mg/kg RLS-0071 on days 21, 22 and 23, and animals receiving rescue doses of 160mg/kg RLS-0071 on days 22 and 23. Asthma animals groups were sacrificed on day 20-24, asthmatic rats receiving RLS-0071 were sacrificed on day 24, BALF fluid was collected, and total protein levels were determined by BCA protein assay. Data are mean and standard error of mean.

FIG. 30 shows that RLS-0071 reduces free MPO levels in BALF in asthmatic rats. The following experimental groups were evaluated: sham animals (unstimulated), animals receiving OVA/LPS regimen (asthma), asthmatic animals receiving prophylactic doses of 160mg/kg RLS-0071 on days 21, 22 and 23, and animals receiving rescue doses of 160mg/kg RLS-0071 on days 22 and 23. Asthma rats were sacrificed on day 20-24, asthma rats receiving RLS-0071 were sacrificed on day 24, BALF fluid was collected, and MPO levels were determined by colorimetric assay. Data are mean and standard error of mean. * P=0.05, compared to asthmatic animals (day 24).

FIG. 31 shows that RLS-0071 reduces free DNA levels in BALF in asthmatic rats. The following experimental groups were evaluated: sham animals (unstimulated), animals receiving OVA/LPS regimen (asthma), asthmatic animals receiving prophylactic doses of 160mg/kg RLS-0071 on days 21, 22 and 23, and animals receiving rescue doses of 160mg/kg RLS-0071 on days 22 and 23. Asthma rats were sacrificed on day 20-24, asthma rats receiving RLS-0071 were sacrificed on day 24, BALF fluid was collected, and free DNA levels were determined by the PicoGreen assay. Data are mean and standard error of mean.

FIG. 32 shows that RLS-0071 binds to human VEGF in a dose dependent manner. VEGF was coated on microtiter plates and incubated with increasing concentrations of RLS-0071 followed by detection using antibodies to the peptide, followed by a secondary antibody-HRP conjugate. The signal generated from the HRP conjugate was then read in a plate reader at an OD of 450 nm. C1q was used as positive control for binding.

FIG. 33 shows that RLS-0088 binds to human VEGF at low levels. VEGF was coated on microtiter plates and incubated with 1mg/ml RLS-0071 (positive control) or RLS-0088. The peptides were then detected using antibodies against the peptides, followed by a secondary antibody-HRP conjugate. The signal generated from the HRP conjugate was then read in a plate reader at an OD of 450 nm.

FIG. 34 shows that RLS-0071 and RLS-0088 inhibit VEGF binding to VEGFR-2 and cell signaling. To assess the ability of RLS-0071 and RLS-0088 to inhibit VEGF signaling, the Promega VEGF bioassay was used. This cell-based bioluminescence assay uses luciferase as a read-out to measure binding of VEGF to VEGFR-2 on the recipient cells. Bioluminescence signal was using Bio-Glo ^TM Luciferase assay systems and standard luminometers. Increasing concentrations of VEGF resulted in dose-dependent increases in luminescence (positive control, diamonds). Cells were incubated with increasing concentrations of peptide and then stimulated with human VEGF (black lines show levels of VEGF stimulation alone as a reference). Both RLS-0071 and RLS-0088 inhibited VEGF-mediated signaling in a dose-dependent manner (square and triangle, respectively).

FIG. 35.RLS-0071 and RLS-0088 inhibit angiogenesis in Human Umbilical Vein Endothelial Cells (HUVEC) 3-dimensional culture systems. Purified HUVECs were stained with CellTrace dye, preincubated with RLS-0071 and RLS-0088, and then added to LPS-containing extracellular matrix to stimulate angiogenesis. The cells were then incubated overnight at 37 ℃ and angiogenesis (formation of neoducts and budding) was observed by visualization on an inverted microscope.

FIG. 36 RLS-0071 inhibited angiogenesis in HUVEC basement membrane mediated culture systems. Purified HUVEC was pre-incubated with increasing concentrations of RLS-0071 for 30min. Cells were applied to the basement membrane matrix layer containing LPS to stimulate angiogenesis and incubated at 37 ℃ for 18 hours. Angiogenesis (formation and budding of the neovasculature) was observed by light microscopy.

Detailed Description

As described in the background section, there is a great need in the art for techniques to identify peptide-based inhibitors for different pathways of the complement system and to exploit this understanding to develop new therapeutic peptides. The present invention meets this and other needs. Embodiments of the present invention relate generally to synthetic peptides, and more particularly to pegylated forms of the synthetic peptides.

In order to facilitate an understanding of the principles and features of various embodiments of the present invention, various illustrative embodiments are explained below. Although exemplary embodiments of the present invention are explained in detail, it should be understood that other embodiments are also contemplated. Accordingly, it is not intended that the scope of the invention be limited to the details of construction and arrangement of the components set forth in the following description or examples. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Furthermore, in describing exemplary embodiments, specific terminology will be resorted to for the sake of clarity.

It must also be noted that, as used in this specification and the appended claims, no particular number of a reference includes a plurality of reference unless the context clearly dictates otherwise. For example, references to components are also intended to include compositions of multiple components. References to compositions containing "ingredients" are intended to include other ingredients in addition to the ingredients mentioned. In other words, a reference to a specific number does not denote a limitation of quantity, but rather indicates that there is "at least one of the referenced item.

As used herein, the term "and/or" may mean "and," may mean "or," may mean "exclusive or," may mean "one," may mean "some but not all," may mean "neither," and/or may mean "both. The term "or" is intended to mean an inclusive "or".

Furthermore, in describing exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term take into account its broadest meaning as understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to accomplish a similar purpose. It is understood that embodiments of the disclosed technology may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description. References to "one embodiment," "an example embodiment," "some embodiments," "certain embodiments," "various embodiments," etc., indicate that the embodiment of the disclosed technology so described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Furthermore, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

As used herein, the term "about" should be interpreted to mean both numbers specified as an end point of any range. Any reference to a range should be considered as providing support for any subset within that range. Ranges may be expressed herein as from "about" or "approximately" or "substantially" one particular value, and/or to "about" or "approximately" or "substantially" another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value to the other particular value. Furthermore, the term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, the error range depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within an acceptable standard deviation in accordance with the practice in the art. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, more preferably up to ±1% of a given value. Alternatively, the term may mean within an order of magnitude, preferably within a factor of 2, of a value, particularly for biological systems or processes. Where a particular value is described in the application and claims, unless otherwise stated, the term "about" is implicit and in this case means within an acceptable error range for the particular value.

Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the present invention. Accordingly, the description of a range should be considered to specifically disclose all possible sub-ranges and individual values within the range. For example, descriptions of ranges such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values within the ranges such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

Likewise, as used herein, a "substantially free of" something or "substantially pure" or the like can include both "at least substantially free of" something or "at least substantially pure" and "completely free of" something or "completely pure".

"comprising" or "containing" or "including" means that at least the recited compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, materials, particles, method steps have the same function as the recited substance.

Throughout this specification, various components may be identified as having particular values or parameters, however, these entries are provided as exemplary embodiments. Indeed, the exemplary embodiments are not limited to the various aspects and concepts of the invention, as many comparable parameters, dimensions, ranges, and/or values may be implemented. The terms "first," "second," and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

It should be noted that terms like "specifically," "preferably," "typically," "generally," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention. It should also be noted that terms such as "substantially" and "about" are utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation.

Dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Rather, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "50mm" is intended to mean "about 50mm".

It should also be understood that reference to one or more method steps does not exclude the presence of additional method steps or intervening method steps between those specifically identified. Likewise, it should also be understood that reference to one or more components in a composition does not preclude the presence of additional components than those explicitly identified.

The materials constituting the various elements of the invention described below are intended to be illustrative and not limiting. Many suitable materials that perform the same or similar functions as the materials described herein are intended to be included within the scope of the present invention. Such other materials not described herein may include, but are not limited to, materials developed after the time of development of the present invention, for example. Any dimensions listed in the various figures are for illustration purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and are intended to be included within the scope of the present invention.

As used herein, the term "subject" or "patient" refers to a mammal and includes, but is not limited to, humans and veterinary animals. In a preferred embodiment, the subject is a human.

As used herein, the term "combination" of a synthetic peptide according to the claimed invention with at least a second pharmaceutically active ingredient means any desired combination of at least two compounds that can be delivered simultaneously or sequentially (e.g., over a 24 hour period). It is contemplated that the compositions and methods of the present invention, when used to treat a variety of different diseases, may be used with other therapeutic methods/agents suitable for the same or similar diseases. Such other therapeutic methods/agents may be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. Due to additive or synergistic effects, the appropriate therapeutically effective dose of each agent may be reduced.

A "disease" is a state of health of a subject, wherein the subject is unable to maintain homeostasis, and wherein the subject's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in a subject is a state of health, wherein the subject is able to maintain steady state, but wherein the subject's state of health is inferior to the state of health in the absence of the disorder. If untreated, the disorder does not necessarily cause a further decline in the health status of the subject.

The term "treatment" of a condition, disorder or disorder includes: (1) Preventing or delaying the occurrence of at least one clinical or subclinical symptom of the state, disorder or condition in a subject likely to suffer from or susceptible to, but not yet experiencing or exhibiting the clinical or subclinical symptom of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., stopping, alleviating or delaying the progression of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing a decline in the state, disorder or condition or at least one clinical or subclinical symptom thereof. The benefit to the subject to be treated is statistically significant or at least perceptible to the patient or physician.

The term "treatment" as used herein means treatment and/or prevention. Therapeutic effects are obtained by inhibition, reduction, alleviation or eradication of the disease state.

As used herein, the term "therapeutically effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition that is sufficient to effect such treatment when administered to a subject to treat (e.g., prevent or ameliorate) a state, disorder or condition. The "therapeutically effective amount" will vary with the compound or bacteria or analog being administered, the disease and its severity, and the age, weight, physical condition and responsiveness of the mammal to be treated.

The phrase "pharmaceutically acceptable" when used in connection with the compositions of the present invention means that the molecular entities and other ingredients of such compositions are physiologically tolerable and generally do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

The term "pharmaceutical carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Aqueous or aqueous solutions saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the pharmaceutical carrier may be a solid dosage form carrier including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent. Suitable pharmaceutical carriers are described in Remington pharmaceutical (Remington's Pharmaceutical Sciences) of e.w. martin.

The term "analog" or "functional analog" refers to a related modified form of a polypeptide in which at least one amino acid substitution, deletion, or addition is made such that the analog retains substantially the same biological function as the unmodified form in vivo and/or in vitro.

The terms "sequence identity" and "percent identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid to obtain optimal alignment with a second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at the corresponding amino acid or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in a second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity% = number of identical positions/total number of positions (i.e., overlapping positions) ×100). Preferably, the two sequences have the same length.

Several different computer programs are available for determining the degree of identity between two sequences. For example, the comparison of sequences and the determination of percent identity between two sequences may be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (j. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in Accelrys GCG software package (available at www.accelrys.com/products/GCG), using the Blosum 62 matrix or PAM250 matrix, and a GAP weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. These different parameters will produce slightly different results, but the overall percent identity of the two sequences does not change significantly when different algorithms are used.

Sequence comparison may be performed over the entire length of the two sequences that are proportional or within a fragment of the two sequences. Typically, the comparison will be made over the full length of the two sequences, which are proportionally compared. However, sequence identity may be performed over a region of, for example, 20, 50, 100 or more consecutive amino acid residues.

As known in the art, "sequence identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., a reference sequence, and a given sequence to be compared to the reference sequence. Sequence identity is determined by comparing the given sequence to a reference sequence after optimal alignment of the sequences to produce the highest degree of sequence similarity, which is determined by matching between these sequence strings. After such alignment, sequence identity is determined on a position-by-position basis, e.g., a sequence is "identical" at a particular position if the nucleotide or amino acid residues are identical at that position. The total number of such positional identities is then divided by the total number of nucleotides or residues in the reference sequence, giving% sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to the methods described in the following documents: computing molecular biology (Computational Molecular Biology), lesk, a.n. master, oxford University Press, new York (1988); biological calculation: informatics and genome project (Biocomputing: informatics and Genome Projects), smith, d.w. master, academic Press, new York (1993); computer analysis of sequence data (Computer Analysis of Sequence Data), part I, griffin, a.m. and Griffin, h.g. master, humana Press, new Jersey (1994); sequence analysis in molecular biology (Sequence Analysis in Molecular Biology), von Heinge, g., academic Press (1987); sequence analysis primer (Sequence Analysis Primer), grisskov, M. And Devereux, J. Main, M.Stockton Press, new York (1991); and Carilo, H.and Lipman, D., SIAM J.applied Math.,48:1073 (1988), the teachings of which are incorporated herein by reference. The preferred method of determining sequence identity is designed to give the greatest match between the sequences tested. The method of determining sequence identity is encoded in a publicly available computer program that determines sequence identity between given sequences. Examples of such procedures include, but are not limited to, GCG package (Devereux, J. Et al, nucleic Acids Research,12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al, J.molecular. Biol.,215:403-410 (1990)). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, altschul, S.et al, NCBI NLM NIH Bethesda, md.20894; altschul, S.F. et al, J.molecular.biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights to produce the highest level of sequence identity between a given sequence and a reference sequence. By a polynucleotide having, for example, at least 95%, for example at least 96%, 97%, 98%, 99% or 100% "sequence identity" to a reference nucleotide sequence, it is meant that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5, 4, 3, 2, 1 or 0 point mutations in every 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having, for example, at least 95%, for example at least 96%, 97%, 98%, 99% or 100% sequence identity in the nucleotide sequence relative to a reference nucleotide sequence, up to 5%, 4%, 3%, 2%, 1% or 0% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide, or up to 5%, 4%, 3%, 2%, 1% or 0% of the total nucleotides in the reference sequence may be incorporated in the reference sequence. These mutations of the reference sequence may occur at the 5 'or 3' end positions of the reference nucleotide sequence or anywhere between those end positions, interspersed either individually between nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence. Similarly, a polypeptide has a given amino acid sequence that has, for example, at least 95%, for example, at least 96%, 97%, 98%, 99% or 100% sequence identity to a reference amino acid sequence, meaning that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5, 4, 3, 2, 1 or 0 amino acid changes in every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95%, e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity to a reference amino acid sequence, up to 5%, 4%, 3%, 2%, 1% or 0% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or up to 5%, 4%, 3%, 2%, 1% or 0% of the total number of amino acid residues in the reference sequence may be inserted in the reference sequence. These changes in the reference sequence may occur at amino or carboxy terminal positions of the reference amino acid sequence or at any position between those terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. Preferably, the different residue positions differ by conservative amino acid substitutions. However, conservative substitutions are not included as matches when determining sequence identity.

The term "immune response" as used herein includes an innate immune response, a T-cell mediated immune response, and/or a B-cell mediated immune response. Exemplary immune responses include T cell responses such as cytokine production and cytotoxicity, and B cell responses such as antibody production. In addition, the term "immune response" includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes such as B cells and T cells (cd4+, cd8+, th1 and Th2 cells), antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes), natural killer cells, myeloid cells such as macrophages, eosinophils, mast cells, basophils, and granulocytes (e.g., neutrophils).

"parenteral" administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intradermal (i.d.) injection or infusion techniques.

In the context of the medical field, the term "prevention" encompasses any action that reduces the burden of death or morbidity from the disease. Prevention can be performed at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of disease, secondary and tertiary prevention levels encompass activities aimed at preventing disease progression and symptoms from occurring, as well as reducing the negative impact of established disease by restoring function and reducing complications associated with the disease.

"variants" of a polypeptide according to the invention may be: (i) Wherein one or more amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and these replaced amino acid residues may or may not be variants of the residues encoded by the genetic code; (ii) Variants in which there are one or more modified amino acid residues, e.g. residues modified by attachment of a substituent group; (iii) Wherein the polypeptide is a variant of an alternative splice variant of the polypeptide of the invention; (iv) a fragment of said polypeptide; and/or (v) variants in which the polypeptide is fused to another polypeptide, such as a leader or secretory sequence or a sequence for purification (e.g., his tag) or detection (e.g., sv5 epitope tag). Such fragments include polypeptides produced by proteolytic cleavage of the original sequence, including multi-site proteolysis. Variants may be post-translationally or chemically modified. Such variations are considered to be within the scope of those skilled in the art from the teachings herein.

Within the meaning of the present invention, the term "co-administration" is used to refer to simultaneous administration of a composition according to the invention and another therapeutic agent in one composition, or simultaneous administration in a different composition, or sequential (preferably over a 24 hour period).

Conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art can be used in accordance with the invention. Such techniques are well explained in the literature. See, e.g., sambrook, fritsch & Maniatis, guide to molecular cloning experiments (Molecular Cloning: A Laboratory Manual), second edition (1989), cold Spring Harbor Laboratory Press, cold Spring Harbor, new York (herein "Sambrook et al, 1989"); DNA Cloning methods (DNA Cloning: A Practical Approach), volumes I and II (D.N.Glover Main, 1985); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait Main plaited, 1984); nucleic acid hybridization (Nucleic Acid Hybridization) (B.D.Hames & S.J.Higgins, main plaited, 1985); transcription and translation (Transcription and Translation) (b.d. hames & s.j. Higgins, maing., 1984); animal cell culture (Animal Cell Culture) (R.I. Freshney, main code, 1986); immobilized cells and enzymes (Immobilized Cells and Enzymes) (IRL Press, 1986); perbal, molecular cloning Utility guidelines (A Practical Guide To Molecular Cloning) (1984); m.ausubel et al, modern methods of molecular biology (Current Protocols in Molecular Biology), john Wiley & Sons, inc. (1994); etc.

Peptide compositions of the invention

Modification of the amino acid structure of CP1 led to the discovery of other peptides capable of modulating complement activation, e.g., C1q activity. Modifications such as PEGylation have previously been demonstrated to enhance peptide solubility compared to the parent molecule (IALILEPICCQERAA; SEQ ID NO: 1) and to effectively inhibit biological activity in vitro assays of classical complement pathway activation/inhibition, myeloperoxidase (MPO) inhibition, antioxidant activity and NET activity inhibition. Peptides with a monodisperse 24-mer PEGylation moiety at the C-terminus were found to be highly soluble and have strong inhibition of the complement system (IALILEPICCQERAA-dPEG 24; SEQ ID NO:2; PA-DPEG24; PA-dPEG 24). Another suitable peptide is set forth in SEQ ID NO:2 comprises a sarcosine substitution (IALILEP (Sar) CCQERAA; SEQ ID NO:3; PA-I8Sar; RLS-0088).

As used herein, the term "peptide" refers to a peptide based on SEQ ID NO:2, which may be naturally occurring, or may be a peptidomimetic, a peptide analogue, and/or a synthetic derivative (including, for example, but not limited to, a pegylated peptide). In addition, the peptide may have less than about 15 amino acid residues, such as about 10 to about 15 amino acid residues, such as 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 possible peptides in the context of the present invention. Peptides may also have more than 15 amino acids, for example 16, 17, 18, 19 and 20 or more amino acids.

The disclosed peptides are generally constrained (i.e., having certain structural elements, such as amino acids that exist in the initial β -turn or β -sheet layers, or are cyclized, such as by the presence of Cys residues that form disulfide bonds) or unconstrained (i.e., linear) amino acid sequences of greater than about 15 amino acid residues, or less than about 15 amino acid residues.

Substitutions of amino acids within the peptide sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, phenylalanine, tryptophan, and methionine. Amino acids containing an aromatic ring structure include phenylalanine, tryptophan, and tyrosine. Polar central amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. For example, one or more amino acid residues within the sequence may be replaced with another amino acid of similar polarity, which acts as a functional equivalent, producing a silent change.

Conservative changes generally result in less structural and functional changes in the resulting protein. Non-conservative changes are more likely to alter the structure, activity or function of the resulting protein. For example, the peptides of the present disclosure comprise one or more of the following conservative amino acid substitutions: substitution of an aliphatic amino acid such as alanine, valine, leucine and isoleucine with another aliphatic amino acid; serine is replaced by threonine; threonine is replaced by serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of an amide-bearing residue, such as asparagine and glutamine, with another amide-bearing residue; exchanging basic residues such as lysine and arginine for another basic residue; and replacing aromatic residues such as phenylalanine and tyrosine with another aromatic residue.

Particularly preferred amino acid substitutions include:

a) Ala replaces Glu or vice versa so that negative charge can be reduced;

b) Lys replaces Arg or vice versa so that a positive charge can be maintained;

c) Ala replaces Arg or vice versa so that the positive charge can be reduced;

d) Glu replaces Asp or vice versa so that a negative charge can be maintained;

e) Ser replaces Thr or vice versa so that free-OH can be maintained;

f) Gln replaces Asn or vice versa so that free NH can be maintained ₂ ；

g) Lie replaces Leu or Val or vice versa as approximately equivalent hydrophobic amino acids;

h) Phe replaces Tyr or vice versa as an approximately equivalent aromatic amino acid; and

i) Ala replaces Cys or vice versa in order to affect disulfide bond formation.

Substitutions of amino acids within the peptide sequence may be selected from any amino acid including, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine, N-formyl-L-methionine, sarcosine, or other N-methylated amino acids. In certain embodiments, the amino acids within the peptide sequence are replaced with sarcosine. In certain embodiments, the sarcosine residue replaces SEQ ID NO:2, an isoleucine residue at position 8 of 2.

In one embodiment, the invention discloses a synthetic peptide derived from human astrovirus coat protein, said peptide comprising the amino acid sequence of SEQ ID NO:2 and/or 3, and modifications.

TABLE 1 list of peptides of the invention

SEQ ID NO.	Sequence(s)	Description of the invention
			1	IALILEPICCQERAA	PA(PIC1)
2	IALILEPICCQERAA-PEG24	PA-dPEG24
			3	IALILEP(Sar)CCQERAA	PA-I8Sar

In other embodiments, the synthetic peptide is capable of altering cytokine expression, including but not limited to models of Acute Lung Injury (ALI). In certain embodiments, the invention provides a method of altering cytokine expression, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3. In certain embodiments, the synthetic peptide is capable of treating and/or preventing ALI and/or ARDS. In certain embodiments, the synthetic peptide is capable of treating an ocular disease or disorder and asthma. In certain embodiments, the synthetic peptide is capable of modulating angiogenesis.

In other embodiments, the synthetic peptide is capable of inhibiting or altering neutrophil binding and/or adhesion. In certain embodiments, the invention provides a method of inhibiting or altering neutrophil binding and/or adhesion, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In other embodiments, the synthetic peptide is capable of increasing neutrophil survival. In certain embodiments, the invention provides a method of increasing neutrophil survival, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

In other embodiments, the synthetic peptide may bind to a cell surface receptor such as, but not limited to, integrin and/or ICAM in vivo. In certain embodiments, the invention provides a method of inhibiting or altering the binding of neutrophils to a cell surface receptor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

The disclosed peptides can selectively modulate C1q and MBL activation without affecting alternative pathway activity and are therefore ideal for the prevention and treatment of diseases mediated by deregulated activation of the classical and lectin pathways. Specific blockade of the classical and lectin pathways is particularly desirable because both pathways are associated with ischemia reperfusion-induced injury in many animal models [ Castellano et al, "therapeutic targeting of the complement classical and lectin pathways protects against ischemia reperfusion-induced kidney injury" (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 immune complex generated during intestinal ischemia/reperfusion injury" (Early complement factors in the local tissue immunocomplex generated during intestinal ischemia/reperfusion injury), mol. Immunol.2010february;47 (5) 972-81; tjernberg et al, "Acute antibody mediated complement activation mediated lysis of islet cells and may lead to tissue loss in clinical islet transplantation" (act anti-body-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet transplantation), transformation.20088 apr.27;85 1193-9; zhang et al, "role of Natural IgM in myocardial ischemia reperfusion injury" (The role of natural IgM in myocardial ischemia-reperfusion injury), J Mol Cell Cardiol.2006July;41 (1):62-7). Alternative pathways are essential for immune surveillance against invading pathogens, and humans with defects in alternative pathways suffer from severe bacterial infections. By binding and inactivating C1q and MBL, the peptides can efficiently modulate activation of the classical and lectin pathways, while leaving the alternative pathways intact.

As used herein, the term "modulating" refers to: i) Controlling, reducing, inhibiting or modulating the biological function of an enzyme, protein, peptide, factor, byproduct or derivative thereof, alone or in a complex; ii) reducing the amount of a biological protein, peptide or derivative thereof in vivo or in vitro; or iii) interrupt a biological chain known to contain an associated series of events, cascades or pathways of biological or chemical reactions. Thus, for example, the term "modulate" may be used to describe, for example, reducing the amount of an individual component of the complement cascade, reducing the rate or total amount of formation of a component or component complex, or reducing the overall activity of a complex process or series of biological reactions, as compared to a control sample, thereby resulting in a result such as cell lysis, formation of invertase, formation of a complement-derived membrane attack complex, inflammation, or inflammatory disease. In an in vitro assay, the term "modulation" may refer to a measurable change or decrease in certain biological or chemical events, but one of ordinary skill in the art will recognize that the measurable change or decrease is not necessarily all "modulating.

In certain embodiments, the invention relates to therapeutically active peptides having effects of modulating the complement system.

Pharmaceutical compositions of the invention

The present invention provides a pharmaceutical composition capable of modulating the complement system comprising at least one peptide as discussed above and at least one pharmaceutically acceptable carrier, diluent, stabilizer or excipient. The pharmaceutically acceptable carrier, excipient or stabilizer is non-toxic to the recipient at the dosage and concentration used. They may be solid, semi-solid or liquid. The pharmaceutical compositions of the present invention may take the form of tablets, pills, powders, troches, sachets, cachets, elixirs, suspensions, emulsions, solutions or syrups.

The pharmaceutical compositions of the invention are prepared by mixing a peptide of suitable purity with a pharmaceutically acceptable carrier, diluent or excipient. 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 both prophylactic and therapeutic agents for a variety of different disorders and diseases as set forth above. In one embodiment, the composition comprises a therapeutically effective amount of the peptide. In another embodiment, the composition comprises at least one additional active ingredient effective to modulate the complement system. In another embodiment, the composition comprises at least one additional active ingredient effective to treat at least one disease associated with the complement system. In another embodiment, the composition comprises at least one additional active ingredient effective to treat at least one disease not associated with the complement system. As used herein, the term "therapeutically effective amount" refers to the total amount of each active ingredient sufficient to exhibit a benefit to the subject.

The therapeutically effective amount of the peptide will vary depending on several factors, such as the disorder being treated, the severity of the disorder, the time of administration, the route of administration, the rate of excretion of the peptide used, the duration of the treatment, the co-therapy involved, and the age, sex, weight and condition of the subject, etc. The therapeutically effective amount can be determined by one of ordinary skill in the art. Thus, one of ordinary skill in the art may need to titrate the dose and modify the route of administration to obtain maximum therapeutic effect.

Effective daily dosages typically range from about 0.001 to about 200 milligrams per kilogram (mg/kg) of body weight, including from about 5 to about 160mg/kg, from about 10 to about 160mg/kg, from about 40mg/kg to about 160mg/kg, and from about 40mg/kg to about 100mg/kg. Such doses may be achieved by a 1-6 daily dosing regimen. Alternatively, optimal treatment may be achieved by sustained release formulations using less frequent dosing regimens. In certain embodiments, SEQ ID NO:2 and/or 3 is from about 10mg/kg to about 160mg/kg. In certain embodiments, SEQ ID NO:2 and/or 3 is from about 20mg/kg to about 160mg/kg. In certain embodiments, SEQ ID NO:2 and/or 3 is about 40mg/kg to about 160mg/kg. In certain embodiments, a therapeutically effective amount of SEQ ID NO:2 and/or 3 in at least one dose, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3. In certain embodiments, the administration comprises a therapeutically effective amount of SEQ ID NO:2 and/or 3 comprising from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3. In certain embodiments, a therapeutically effective amount of SEQ ID NO:2 and/or 3 in two doses, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3, and the second dose comprises from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3. In certain embodiments, the second dose is administered 30 seconds to 3 hours after the administration of the first dose.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO:2 and/or 3 and at least one pharmaceutically acceptable carrier, diluent or excipient.

The compositions of the present invention may comprise a carrier and/or excipient. Although the peptide of the invention may be used as such for treatment, it may be preferred to administer it in a pharmaceutical formulation, for example in admixture with suitable pharmaceutical excipients and/or carriers selected in accordance with the intended route of administration and standard pharmaceutical practice. The excipient and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients and carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington pharmaceutical sciences and practices (Remington: the Science and Practice of Pharmacy), lippincott Williams & Wilkins (main editions of a.r. gennaro, 2005). The choice of pharmaceutical excipients and carriers can be selected according to the intended route of administration and standard pharmaceutical practice. Oral formulations are readily adaptable to other mixtures, such as milk, yogurt and infant formulas. Solid dosage forms for oral administration may also be used, which may include, for example, capsules, tablets, caplets, pills, troches, lozenges, powders and granules. Non-limiting examples of suitable excipients include, for example, diluents, buffers (e.g., sodium bicarbonate), preservatives, stabilizers, binders, compactors, lubricants, dispersion enhancing agents, disintegrants, antioxidants, flavoring agents, sweeteners, and colorants. Those skilled in the art will be well able to prepare suitable solutions.

In one embodiment of any of the compositions of the invention, the composition is formulated for delivery by a route such as oral, topical, rectal, mucosal, sublingual, nasal/oral gastric feeding tube, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal and intratracheal administration. In one embodiment of any of the compositions of the present invention, the composition takes the form of a liquid, foam, cream, spray, powder or gel. In one embodiment of any of the compositions of the present invention, the composition comprises a buffer (e.g., sodium bicarbonate).

In the methods of the invention, administration of the peptides and compositions may be accomplished by any method known in the art. Non-limiting examples of useful delivery routes include oral, rectal, fecal (by enema) and nasal/oral gastric feeding tubes, as well as parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal and intratracheal administration. The active agent may be systemic after administration or may be localized by use of local administration, intra-wall (intra-muramic) administration, or use of an implant for retaining the active agent at the implantation site.

The useful dosage of the compounds and formulations of the present invention can vary widely depending upon the nature of the disease, the patient's history, the frequency of administration, the mode of administration, the clearance of the agent from the host, and the like. The initial dose may be larger and then a smaller maintenance dose is used. The agent may be administered at low frequency to weekly or biweekly, or may be divided into smaller doses and administered daily, every half week, etc. to maintain an effective dosage level. It is contemplated that a variety of different agents may be effective in achieving a therapeutic effect. Although the compound of the invention may be used as such for treatment, it may be preferred to administer it in a pharmaceutical formulation, for example in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient diluent and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients, diluents and carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington pharmaceutical sciences and practices (Remington: the Science and Practice of Pharmacy), lippincott Williams & Wilkins (a.r.gennaro master, 2005). The choice of pharmaceutical excipients, diluents and carriers may be selected according to the intended route of administration and standard pharmaceutical practice.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

The solution or suspension may comprise any combination of any of the following components: sterile diluents, including for example, but not limited to, water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetate, citrate and phosphate; and agents for regulating tonicity, such as sodium chloride or dextrose.

In cases where the agent exhibits insufficient solubility, a method of solubilizing the agent may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as 80, or dissolved in aqueous sodium bicarbonate. Pharmaceutically acceptable derivatives of the agents may also be used to formulate effective pharmaceutical compositions.

Together with the active agent, the composition may also contain, for example, but not limited to: diluents, such as lactose, sucrose, dicalcium phosphate or carboxymethyl cellulose; lubricants, such as magnesium stearate, calcium stearate, and talc; and binders such as starch, natural gums such as acacia, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose and its derivatives, povidone, crospovidone and other such binders known to those skilled in the art. Liquid pharmaceutically acceptable compositions may be prepared, for example, by dissolving, dispersing or otherwise mixing an active agent as defined above and optionally a pharmaceutical adjuvant in a carrier such as, but not limited to, water, saline, aqueous dextrose, glycerol, ethylene glycol, ethanol, and the like, to form a solution or suspension. If desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting, emulsifying or solubilizing agents, pH buffering agents and the like, such as, but not limited to, acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate and other such agents. Practical methods of preparing such dosage forms are known or will be apparent to those skilled in the art (e.g., remington's Pharmaceutical Sciences, mack Publishing Company, easton, pa., 15 th edition, 1975). In any event, the composition or formulation to be administered will contain an amount of active agent sufficient to alleviate symptoms in the subject being treated.

The active agent or pharmaceutically acceptable derivative may be prepared with a carrier, such as a time release formulation or coating, that protects the agent from rapid elimination from the body. The composition may contain other active agents to achieve the desired combination of properties.

Parenteral administration, typically characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms as liquid solutions or suspensions, solid forms or emulsions suitable for dissolution or suspension in liquid prior to injection. Suitable excipients include, for example, but are not limited to, water, saline, dextrose, glycerol, or ethanol. In addition, the pharmaceutical compositions to be administered may, if desired, also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other such agents, for example sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures, or formulated as solids or gels. Sterile lyophilized powders are prepared by dissolving the agents provided herein or pharmaceutically acceptable derivatives thereof in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffers known to those skilled in the art, typically at a pH of about neutral. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. Typically, the resulting solution may be dispensed into vials for lyophilization. Each vial may contain, for example, but not limited to, a single dose (10-1000 mg, e.g., 100-500 mg) or multiple doses of the agent. The lyophilized powder may be stored under suitable conditions, for example, at about 4 ℃ to room temperature. Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration.

The compositions of the invention, or pharmaceutically acceptable derivatives thereof, may be formulated as aerosols for administration by inhalation or intranasal means, for example (as described for example in US 4,044,126, 4,414,209 and 4,364,923). These formulations may be in the form of aerosols or nebulisers solutions or as ultra-fine powders for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation may, for example, but not limited to, have a diameter of less than about 50 microns, such as less than about 10 microns.

The agents may also be formulated for topical or surface application, for example to the skin and mucous membranes (e.g. intranasally), in the form of nasal solutions, gels, creams and lotions.

Ophthalmic compositions of the present invention

In certain embodiments, the compositions of the present invention are formulated for ocular administration, including, for example, topical, intravitreal, and/or intraocular administration. In certain embodiments, the composition is delivered to the ocular surface, interconnected innervation, conjunctiva, lacrimal gland, or meibomian gland. The composition may take the form of an eye drop, ointment, gel, foam, solution, suspension and/or intraocular implant.

According to one embodiment, the invention also includes a pharmaceutical composition comprising a therapeutically effective amount of the SEQ ID NO:2. such carriers include, for example, those listed herein.

According to one embodiment, the topical formulations containing the active compound may also contain a physiologically compatible vehicle, which may be selected by the person skilled in the art of ophthalmology using conventional criteria. The vehicle may be selected from known ophthalmic vehicles including, but not limited to, saline solutions, aqueous polyethers such as polyethylene glycol, polyethylenes such as polyvinyl alcohol and povidone, cellulose derivatives such as methyl cellulose and hydroxypropyl methyl cellulose, petroleum derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, acrylic polymers such as carbopol gel, vegetable fats such as peanut oil and polysaccharides such as dextran, and glycosaminoglycans such as sodium hyaluronate and salts such as sodium chloride and potassium chloride.

According to one embodiment, the ophthalmic composition is advantageously applied topically to the eye, in particular in the form of a solution, suspension, ointment, gel or foam. According to another embodiment, the ophthalmic composition is administered by injection or implantation into the eye, intravitreal or aqueous humor.

The exact pharmaceutical formulation (e.g., ophthalmic composition) used in the methods of the present invention will vary according to a wide range of commercial and scientific criteria. That is, one of skill in the art will recognize that the above-described formulations of the invention described herein may contain other agents.

According to one embodiment, conventional pharmaceutically acceptable excipients and additives known to the person skilled in the art are used for the corresponding ophthalmic compositions, such as the types mentioned below, in particular carriers, stabilizers, solubilizers, tonicity enhancing agents, buffer substances, preservatives, thickeners, complexing agents and other excipients. Examples of such additives and excipients can be found in U.S. Pat. nos. 5,134,124 and 4,906,613. Such compositions are prepared in a manner known per se, for example by mixing the active ingredients with the corresponding excipients and/or additives to form the corresponding ophthalmic compositions. The active ingredient is preferably administered in the form of an eye drop, the active ingredient typically being dissolved in, for example, a carrier. Where appropriate, the solution is adjusted and/or buffered to the desired pH and where appropriate stabilizers, solubilizers or tonicity enhancing agents are added. Preservatives and/or other excipients are added to the ophthalmic composition where appropriate.

The carriers used according to the invention are generally suitable for topical or systemic administration and are, for example, water, mixtures of water and water-miscible solvents such as C1-C7-alkanols, vegetable or mineral oils, which contain 0.5 to 5% by weight of hydroxyethylcellulose, ethyl oleate, carboxymethyl cellulose, polyvinylpyrrolidone and other non-toxic water-soluble polymers for ophthalmic use, for example cellulose derivatives such as methyl cellulose, alkali metal salts of carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl hydroxypropyl cellulose and hydroxypropyl cellulose, acrylic esters or methacrylic esters such as salts of polyacrylic acid or ethyl acrylate, polyacrylamides, natural products such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenan, agar and acacia, starch derivatives such as starch acetate and hydroxypropyl starch, and other synthetic products such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxides, preferably crosslinked polyacrylic acids such as neutral Carbopol, or mixtures of these polymers. Preferred carriers are water, cellulose derivatives such as methyl cellulose, alkali metal salts of carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl hydroxypropyl cellulose and hydroxypropyl cellulose, neutral Carbopol or mixtures thereof.

According to one embodiment, the solubilizer used in the ophthalmic composition of the present invention is, for example, tyloxapol, a fatty acid glycerol poly-lower alkylene glycol ester, a fatty acid poly-lower alkylene glycol ester, polyethylene glycol, glycerol ether or a mixture of these compounds. The amount added is generally sufficient to dissolve the active ingredient. For example, the concentration of the solubilizing agent is 0.1 to 5000 times the concentration of the active ingredient. Lower alkylene means a straight or branched chain alkylene having up to and including 7C atoms. Examples are methylene, ethylene, 1, 3-propylene, 1, 2-propylene, 1, 5-pentylene, 2, 5-hexylene or 1, 7-heptylene. The lower alkylene is preferably a straight or branched alkylene having up to and including 4C atoms.

Examples of buffer substances are acetate, ascorbate, borate, bicarbonate/carbonate, citrate, gluconate, lactate, phosphate, propionate and TRIS (tromethamine) buffers. Tromethamine and borate buffers are preferred buffers. For example, the amount of buffer substance added is that amount necessary, for example, to ensure and maintain a physiologically tolerable pH range. The pH range is typically in the range of 5 to 9, preferably 6 to 8.2, more preferably 6.8 to 8.1.

The tonicity enhancing agent is, for example, an ionic compound, such as an alkali or alkaline earth metal halide, such as CaCl ₂ KBr, KCl, liCl, naBr, naCl or boric acid. Nonionic tonicity enhancing agents are, for example, urea, glycerin, sorbitol, mannitol, propylene glycol or dextrose. For example, sufficient tonicity enhancing agent is added to impart an osmolality of about 50 to 1000mOsmol, preferably 100 to 400mOsmol, more preferably 200 to 400mOsmol, even more preferably 280 to 350mOsmol to the ready-to-use ophthalmic composition.

Examples of preservatives are quaternary ammonium salts such as cetrimide, benzalkonium chloride or benzoonium chloride, alkyl mercury salts of thiosalicylic acid such as thimerosal, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate, parabens such as methylparaben or propylparaben, alcohols such as chlorobutanol, benzyl alcohol or phenylethanol, guanidine derivatives such as chlorhexidine or polyhexamethylene biguanide, or sorbic acid. Preferred preservatives are cetrimide, benzalkonium chloride, benzoonium chloride and parabens. Where appropriate, a sufficient amount of preservative is added to the ophthalmic composition to ensure protection against secondary contamination by bacteria and fungi during use.

According to one embodiment, the ophthalmic composition may comprise other non-toxic excipients, such as emulsifiers, wetting agents or fillers, for example polyethylene glycols denoted 200, 300, 400 and 600 or carbowaxes denoted 1000, 1500, 4000, 6000 and 10000. Other excipients that may be used if desired are listed below, but they are not intended to limit the scope of possible excipients in any way. They are in particular complexing agents such as disodium EDTA or EDTA, antioxidants such as ascorbic acid, acetylcysteine, cysteine, sodium bisulphite, butylhydroxyanisole, butylhydroxytoluene or alpha-tocopherol acetate, stabilizers such as cyclodextrin, thiourea, thiosorbitol, sodium dioctyl sulfosuccinate or monothioglycerol, or other excipients such as sorbitol laurate, triethanolamine oleate or palmitate. Preferred excipients are complexing agents such as disodium EDTA and stabilizers such as cyclodextrin. The amount and type of excipient added meets specific requirements and is typically in the range of about 0.0001 to about 90 weight percent. Cyclodextrin consists of several glucose units, each glucose having three free hydroxyl groups. The amount of cyclodextrin used according to one embodiment may preferably be in the range of 0.01-20 wt%, more preferably 0.1-15 wt%, even more preferably 1-10 wt%.

According to one embodiment, the present invention also relates to an ophthalmic composition comprising a therapeutically effective amount of the SEQ ID NO: 2. the carrier, solubilising agent and another therapeutically effective agent, which may be, for example, an antibiotic, an antiallergic agent, an anesthetic, another anti-inflammatory agent, a corticosteroid, an agent suitable for lowering intraocular pressure or another drug.

The ophthalmic composition used in the method of the present invention is preferably prepared using a physiological saline solution as a vehicle. The pH of the ophthalmic composition may be maintained at a substantially neutral pH (e.g., about 7.4, in the range of about 6.5 to about 7.4, etc.) with suitable buffer systems known to those skilled in the art (e.g., acetate buffer, citrate buffer, phosphate buffer, borate buffer).

Topical formulations

Ophthalmic ointments tend to contact the active agent with the eye for a longer period of time than suspensions and certain solutions. Most ointments tend to blur vision because they are not easily removed by tears. Thus, ointments are commonly used at night as an adjunct therapy to eye drops used in the daytime.

The oily ointment base of the composition of the invention is a mixture of mineral oil, petrolatum and lanolin, all having a melting point near body temperature. In the case of the compounds of the invention, the composition may comprise mineral oil, petrolatum or lanolin. According to one embodiment, the preferred composition may include a combination of petrolatum, mineral oil and lanolin. Another preferred composition is an ointment composition comprising white petrolatum, mineral oil and lanolin (anhydrous).

Other exemplary topical formulations include eye drops, inserts, eye shields, impregnated contact lenses, pump delivery systems, dimethyl sulfoxide (DMSO) based solutions and/or suspensions, and liposomes.

Eye drops can be prepared by dissolving the active ingredient in a sterile aqueous solution such as physiological saline, buffer solution, etc., or by incorporating the powder composition to be dissolved prior to use. As is known in the art, other vehicles may be selected, including but not limited to balanced salt solutions, saline solutions, water soluble polyethers such as polyethylene glycols, polyethylenes such as polyvinyl alcohol and povidone, cellulose derivatives such as methyl cellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, acrylic polymers such as carbopol gel, vegetable fats such as peanut oil, polysaccharides such as dextran, and glycosaminoglycans such as sodium hyaluronate. Additives commonly used in eye drops may be added if desired. Such additives include isotonic agents (e.g., sodium chloride, etc.), buffering agents (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), thickening agents (e.g., sugars such as lactose, mannitol, maltose, etc.), e.g., hyaluronic acid or salts thereof such as sodium hyaluronate, potassium hyaluronate, etc., e.g., mucopolysaccharides such as chondroitin sulfate, etc., e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, or other agents known to those skilled in the art.

The solubility of the components of the compositions of the present invention may be enhanced by the surfactant or other suitable co-solvent in the composition. Such co-solvents include polysorbate 20, 60 and 80, pluronic F68, F-84 and P-103, cyclodextrins or other agents known to those skilled in the art. Such co-solvents may be used at levels of about 0.01 wt% to 2 wt%.

The compositions of the present invention may be formulated in sterile unit dosage forms without preservatives. The compositions of the present invention may be packaged in multiple dosage forms. To prevent microbial contamination during use, preservatives may be preferred. Suitable preservatives include benzalkonium chloride, thimerosal, chlorobutanol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, phenethyl alcohol, disodium edetate, sorbic acid, onamer M, or other agents known to those skilled in the art. In prior art ophthalmic products, such preservatives may be used at a level of 0.004% to 0.02%. In the compositions of the present application, the preservative, preferably benzalkonium chloride, may be used at a level of from 0.001% to less than 0.01%, such as from 0.001% to 0.008%, preferably about 0.005% by weight. It has been found that a benzalkonium chloride concentration of 0.005% may be sufficient to protect the compositions of the present invention from microbial attack.

The formulation of the invention may be administered in a few drops, one to four drops, preferably one to three drops, more preferably one to two drops, most preferably one drop per day, per administration. Alternatively, the formulation of the invention may be applied or sprayed several times per day, preferably one to six times per day, more preferably one to four times, most preferably once.

Conjunctiva/sclera formulation

The topical conjunctival access route allows the drug to penetrate into the anterior ocular segment. In addition, it has been shown that topically administered drugs can enter the sclera from the conjunctiva. The sclera has been shown to be readily permeable even to large molecular weight compounds (-150 kD). Comprising SEQ ID NO:2 and/or 3 is a suitable formulation for topical conjunctival and scleral administration. The pharmaceutical composition may also be administered by subconjunctival injection.

Intraocular/intravitreal formulations

The pharmaceutical compositions of the invention may be formulated for intraocular or intravitreal administration by injection (e.g. periocular, subconjunctival, intracameral, intraocular or intravitreal injection) or by introduction of a suitable implant (e.g. intracorneal or intraocular implant).

Implantable formulations

In one embodiment, an implant comprising an ophthalmic composition of the present invention is formulated with PIC1 peptide embedded in a biocompatible, biodegradable/bioerodible polymer matrix. Release of the agent is achieved by erosion of the polymer, followed by exposure of previously entrapped agent particles to the vitreous, and then dissolution and release of the agent. The release kinetics achieved by this form of drug release are different from those achieved by formulations that release drug by polymer swelling (e.g. using hydrogels such as methylcellulose). In the latter case, the drug is released not by polymer erosion, but by polymer swelling, which releases the drug as the liquid diffuses through the exposed path. Parameters that determine the release kinetics include the size of the drug particles, the water solubility of the drug, the ratio of drug to polymer, the method of manufacture, the exposed surface area, and the erosion rate of the polymer.

Diffusion of PIC1 peptide from the implant may also be controlled by the structure of the implant. For example, diffusion of PIC1 peptide from an implant may be controlled using a membrane attached to a polymer layer containing a drug. The membrane layer will be intermediate the polymer layer comprising the peptide and the desired treatment site. The membrane may be composed of any of the biocompatible materials described above, depending on the presence of agents other than the peptides present in the polymer, the composition of the polymer comprising the PIC1 peptide, the desired diffusion rate, etc. For example, the polymer layer typically contains a very large amount of peptide, and is typically saturated. Such PIC1 peptide-saturated polymers typically release the peptide at a very high rate. In this case, the release of the peptide may be slowed by selecting a membrane having a lower permeability for the peptide than the polymer. Because of the lower peptide permeability of the membrane, the peptide will remain concentrated in the polymer and the overall diffusion rate will be determined by the peptide permeability of the membrane. Thus, the release rate of the peptide from the implant is reduced, thereby providing a more controlled and sustained delivery of the peptide to the treatment site.

Ocular administration

Administration of the ophthalmic compositions of the present invention may be by intraocular injection, although other modes of administration may be effective. Typically, the ophthalmic composition will be delivered intra-ocular (via a chemical delivery system or invasive device) to the individual. However, the invention is not limited to intraocular delivery, as it also includes topical (extraocular administration) or systemic (e.g., oral or other parenteral route such as subcutaneous administration) so long as a sufficient amount of peptide within cells or tissues located in or near the eye achieves contact with the site of ocular disease. Parenteral administration is used where appropriate by the physician. Preferably, the ophthalmic composition is administered in unit dosage form suitable for single administration of precise doses.

As mentioned above, in situ delivery to the intraocular region may be achieved by injection, cannula, or other invasive device designed to introduce a precisely metered amount of the desired ophthalmic composition into a specific compartment or tissue (e.g., posterior chamber or retina) within the eye. Intraocular injection may be in the vitreous (intravitreal) or under the conjunctiva (subconjunctival) or behind the eye (retrobulbar), in the sclera or under the terse (subcontracting), and may take the form of a depot. Other intraocular routes of administration and injection sites and forms are also contemplated and within the scope of the invention.

Topical application of the ophthalmic composition of the present invention for the treatment or prevention of ophthalmic disorders may be as an ointment, gel, foam or eye drops. Preferably an osmotic composition comprising PIC1 peptide is used. The topical ophthalmic composition may also be an aqueous formulation that is gellable in situ. Such formulations comprise a gelling agent in a concentration effective to promote gelling upon contact with the eye or tear fluid outside the eye. Suitable gelling agents include, but are not limited to, thermosetting polymers such as tetra-substituted ethylenediamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamines), polycarbophil, and polysaccharides such as gellan gum, carrageenan (e.g., kappa-carrageenan and iota-carrageenan), chitosan and alginate gums.

The amount of PIC1 peptide to be administered and the concentration of the compound in the topical ophthalmic composition used in the method depend on the diluent, the delivery system or selected device, the clinical condition of the patient, side effects, and the stability of the compound in the formulation. Thus, the physician uses a suitable formulation containing the appropriate concentration of peptide and selects the amount of formulation to administer based on clinical experience with the patient or similar patient.

Slow or extended release delivery systems include any of a variety of biopolymers (bio-based systems), systems employing liposomes, colloids, resins, and other polymeric delivery systems or compartmentalized reservoirs, which may be used with the compositions described herein to provide a continuous or long-term source of therapeutic compounds.

The skilled reader will appreciate that the length of time any ophthalmic composition used in the methods of the present invention resides in the ocular environment will depend upon factors such as the physicochemical and/or pharmacological properties of the compound used in the formulation, the concentration of the compound used, the bioavailability of the compound, the disease to be treated, the mode of administration, and the preferred lifetime of the treatment, among others.

The frequency of treatment according to the methods of the invention is determined according to the disease being treated, the deliverable concentration of PIC1 peptide, and the method of delivery. The dosing frequency may be once a month if the peptide is delivered by intravitreal injection. Preferably, the dosing frequency is once every three months. Dose frequency can also be determined by observation and the dose delivered when the previously delivered peptide is significantly cleared. Once the therapeutic result is obtained, the peptide may be gradually reduced or stopped. Occasionally, side effects may require cessation of treatment. In general, an effective amount of the compound is an amount that provides subjective relief of symptoms or an objectively identifiable improvement noted by a clinician or other qualified observer.

Nasal compositions of the invention

In certain embodiments, the compositions of the present invention are formulated for nasal administration, including, for example, inhalation, insufflation, or nebulization. The composition may take the form of, for example, nasal drops, nasal sprays and formulations suitable for inhalation, insufflation and/or nebulization.

According to one embodiment, the invention further comprises a pharmaceutical composition comprising a therapeutically effective amount of the SEQ ID NO:2 and/or 3. Such vectors include, for example, those listed herein.

Nasal administration

Administration of the nasal compositions of the present invention may be by nasal drops, sprays, inhalable and nebulized formulations, although other modes of administration may be effective. However, the invention is not limited to nasal delivery, as it also includes local (intranasal administration) or systemic (e.g., oral or other parenteral route such as subcutaneous administration) so long as a sufficient amount of peptide within the cells or tissues located in the nose achieves therapeutic efficacy. Parenteral administration is used where appropriate by the physician. Preferably, the nasal composition is administered in unit dosage form suitable for single administration of precise doses.

As mentioned above, in situ delivery to the intranasal region may be achieved by a spray, drops, or inhaler or nebulizer device designed to introduce precisely metered amounts of the desired nasal composition into the nasal passages. Other intranasal routes and forms of administration are also contemplated and within the scope of this invention.

The amount of PIC1 peptide to be administered and the concentration of the compound in the nasal composition used in the method depend on the diluent, the delivery system or selected device, the clinical condition of the patient, side effects, and the stability of the compound in the formulation. Thus, the physician uses a suitable formulation containing the appropriate concentration of peptide and selects the amount of formulation to administer based on clinical experience with the patient or similar patient.

The skilled reader will appreciate that the length of time any nasal composition used in the methods of the present invention will remain in the nasal environment will depend upon factors such as the physicochemical and/or pharmacological properties of the compound used in the formulation, the concentration of the compound used, the bioavailability of the compound, the disease to be treated, the mode of administration, and the preferred lifetime of the treatment, among other factors.

The frequency of treatment according to the methods of the invention is determined according to the disease being treated, the deliverable concentration of PIC1 peptide, and the method of delivery. The dosing frequency may be once a month if the peptide is delivered by nasal inhalation or insufflation. Preferably, the dosing frequency is once every three months. Dose frequency can also be determined by observation and the dose delivered when the previously delivered peptide is significantly cleared. Once the therapeutic result is obtained, the peptide may be gradually reduced or stopped. Occasionally, side effects may require cessation of treatment. In general, an effective amount of the compound is an amount that provides subjective relief of symptoms or an objectively identifiable improvement noted by a clinician or other qualified observer.

Combination therapy

Another embodiment of the invention provides a method of modulating the complement system comprising administering to a subject a pharmaceutical composition of the invention. Although the pharmaceutical compositions of the present invention may be administered as the sole active agent, they may also be used in combination with one or more therapeutic or prophylactic agents effective in modulating the complement system. In this aspect, the methods of the invention comprise administering the pharmaceutical compositions of the invention prior to, concurrently with, and/or after one or more other therapeutic or prophylactic agents effective to modulate the complement system.

The pharmaceutical composition of the present invention may be administered together with other agents in combination therapy or separately, or by combining the pharmaceutical composition with the other agents into one composition. The dose is administered and adjusted to achieve maximum modulation of the complement system. For example, both the pharmaceutical composition and the other agent are typically present at a dosage level of between about 10% to about 150%, more preferably between about 10% to about 80%, of the dosage normally administered in a monotherapy regimen.

Examples

The invention is further illustrated and demonstrated by the following examples. However, the use of these and other embodiments anywhere in this specification is illustrative only, and in no way limits the scope and meaning of the invention or any exemplary terms. Likewise, the invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading the present specification, and such variations may be made without departing from the spirit or scope of the invention. The invention is, therefore, to be limited only by the following claims, along with the full scope of equivalents to which such claims are entitled.

Example 1: effects of PA-dPEG24 on cytokine expression in ALI and lung injury

Acute lung injury occurring in severe cases of covd-19 has been demonstrated to be caused by a host immune response involving a cytokine storm (Mehta et al, 2020), and NETosis is presumed to be a key driver of Acute Lung Injury (ALI) in covd-19 patients by direct lung injury and contributing to cytokine production (Barnes et al, 2020). To determine single doses of 10 and 160mg/kg of SEQ ID NO administered prior to infusion: 2 or a single dose of 40 and 160mg/kg of SEQ ID NO:2 effects on cytokine levels, a final blood sample was obtained. Plasma was isolated from blood samples and analyzed for the following inflammatory cytokines by xMAP bead-based immunoassays: IFNγ, IL-6, IL-2, IL-10, TNF α, MCP-1 (CCL-2), RANTES (CCL-5), MIP1 α (CCL-3), IL-1 β and MIP-2 (CXCL 2). This dose of SEQ ID NO:2 tests were performed in a two hit ALI rat model (river et al 2020). In this model, which is a modified model of the established two-hit ALI model (kilman et al, 1997; kilman, 2006), young male Wistar rats were injected with Lipopolysaccharide (LPS) (first hit) to excite neutrophils, and then 30% incompatible erythrocytes (second hit) were infused after 30 minutes to induce complement activation. Most importantly, this two-hit ALI model produces an extremely robust response in a broad range of pro-inflammatory cytokines, replicating the cytokine storm. 10 or 160mg/kg dose administered prior to infusion or 40 or 160mg/kg dose administered after infusion of SEQ ID NO:2 modulating cytokine levels, with a significant decrease or trend in decreased levels of pro-inflammatory cytokines (fig. 1). Other inflammatory cytokines analyzed by xMAP bead-based immunoassays include IL-5, IL-18, IL-1α, IL-13, IL-17, IL-12, and IP-10 (FIG. 2). PA-dPEG24 at either a 10 or 160mg/kg dose administered prior to infusion or a 40 or 160mg/kg dose administered after infusion regulates cytokine levels with a tendency to significantly reduce or decrease the levels of pro-inflammatory cytokines. In the second run, plasma samples from the following groups were analyzed: sham surgery, 1 strike, 2 strikes, 2 strikes+10 mg/kg prophylactic dose of RLS-0071,2 strikes+160 mg/kg prophylactic dose of RLS-0071,2 strikes+40 mg/kg rescue dose of RLS-0071 at 30 seconds, and 2 strikes+30 seconds of 160mg/kg rescue dose of RLS-0071 (FIGS. 16A-16C and 17A-17C). For each cytokine reported, two replicates were run per animal. Data are mean and standard error of mean.

Importantly, in contrast to the reduction of inflammatory Th1 and Th17 cytokines shown in fig. 1 and 2, PA-dPEG24 did not significantly reduce the level of anti-inflammatory Th2 cytokine IL-4 (fig. 3). Th2 cells promote alternative activation of M2 macrophages involved in the reduction of pathological inflammation and counteract Th1 responses.

These findings surprisingly show that PA-dPEG24 can modulate the pro-inflammatory response in ALI. Unexpectedly, PA-dPEG24 can modulate inflammatory cytokine levels in such ALI animal models. The observation that both lung injury and inflammatory cytokine levels, assessed by histology, were reduced by both prophylactic and salvage administration of PA-DPEG24, suggests that this molecule may reduce multiple inflammatory pathways that have an impact on ALI.

In addition to the cytokines shown above, PA-dPEG24 administered at a dose of 10 or 160mg/kg before infusion or at a dose of 40 or 160mg/kg after infusion also regulated other cytokines and growth factors levels, which were significantly reduced or had a tendency to be reduced in level (fig. 4).

RLS-0071 reduces neutrophil mediated ALI

The two-hit ALI model previously developed by the inventors began with LPS infused (first hit) into Wistar rats, then 30% incompatible erythrocytes were infused after 30 minutes (second hit), and animals were sacrificed after 4 hours. The lungs of animals showed significant neutrophil mediated ALI and robust complement activation and NETosis as measured by C5a levels and free DNA in the blood stream, respectively. To assess the ability of RLS-0071 to reduce lung injury in this model, animals were treated with a single prophylactic dose of RLS-0071 administered 2 minutes prior to the second hit or with a rescue dose administered at various times after the second hit. Lungs were isolated from animals 4 hours after the second strike and tissues were assessed by H & E staining. Sham operated animals (fig. 18A) or animals that received only the first hit of LPS (fig. 18B) showed normal lung tissue structure, whereas animals that received 2 hits showed dramatic lung lesions mediated by significant neutrophil infiltration into the alveolar wall (fig. 18C). In contrast, animals that received prophylactic administration of RLS-0071 at 10, 40 or 160mg/kg 2 minutes prior to incompatible erythrocyte infusion showed a significant reduction in lung injury, and lung tissue showed similar lung morphology as sham operated animals (fig. 18D-18F). Animals receiving rescue administration of 40mg/kg RLS-0071 at 0.5, 60, 90, 120 and 180min after the administration of the second hit also showed similar lung tissue structures as sham operated animals (FIGS. 18G-18K).

To determine the level of lung tissue protection of RLS-0071 in this model, the cell wall thickening of H & E sections of different treatment groups was graded. Images of random microscopic fields were turned black and white and quantified by ImageJ (NIH) analysis. The ratio of black to white pixels is then determined as a measure of lung injury: as lung injury increases, the alveolar walls thicken and the alveolar space (white space) contracts, resulting in a decrease in white pixels and an increase in black pixels. Consistent with the lack of tissue damage visualized directly by microscopic observation of H & E sections, sham operated animals and animals receiving only the first hit of LPS had low lung injury scores, whereas animals receiving 2 hits showed much higher injury scores, as previously demonstrated (fig. 19). Animals receiving a prophylactic dose of RLS-0071 at 10mg/kg showed a significant reduction in lung injury (p=0.002) compared to untreated 2-hit animals, and this effect was enhanced in animals receiving a prophylactic dose of 40mg/kg (p < 0.001). Rats given 160mg/kg RLS-0071 prophylactically had similar lung scores to the 40mg/kg dose, indicating that doses exceeding 40mg/kg did not provide any additional protection to lung tissue (p=0.33, comparing doses of 40mg/kg and 160 mg/kg) (fig. 19). To assess whether animals given at various times after the second stroke could alleviate lung injury, animals that underwent two stroke challenges were treated with 40mg/kg RLS-0071 at 0.5, 60, 90, 120 and 180 minutes after erythrocyte infusion. Treatment with RLS-0071 showed significant reduction of lung injury at all time points after the second hit (all p < 0.001) (fig. 19). These results indicate that in this experimental model, a single dose of RLS-0071 can significantly attenuate acute lung injury up to 3 hours after 2 hits.

RLS-0071 reduces C5a production in blood

Animals receiving only the first stroke of LPS showed an increase in C5a levels, which can be attributed to the alternative pathway activation mediated by LPS, whereas animals receiving 2 strokes showed much higher C5a levels due to the combination of alternative pathway activation by LPS with classical pathway-mediated activation by incompatible erythrocyte infusion. To evaluate the effect of RLS-0071 on C5a in this model, rats subjected to 2 challenge shots were treated with prophylactic or rescue doses of RLS-0071 and C5a levels were measured from blood samples taken 0, 5 minutes and 1 hour after the second challenge shot. Sham operated animals had baseline levels of C5a production, whereas animals receiving the first hit of LPS showed increased levels at 5 minutes and 1 hour (figure 20). As expected, animals receiving 2 hits had significantly more C5a production at the 1 hour time point (figure 20). Animals receiving prophylactic doses of 10, 40 and 160mg/kg RLS-0071 showed a significant decrease in C5a at the 5 min time point (p < 0.001) for each dose group, and the decrease in C5a reached a significant decrease at the 1 hr time point (p=0.002) with the 10mg/kg dose except for the 180 min rescue dose (fig. 20). As observed with prophylactic dosing, RLS-0071 dosed at 40mg/kg rescue dose at 0.5 (p=0.001), 60 (p < 0.001), 90 (p < 0.001) and 120 (p=0.001) minutes after 2 challenge at the 5 minute time point showed a significant decrease in C5a levels, except animals receiving the rescue dose at 180 minutes (fig. 20). At the 1 hour time point, all rescue doses had significantly reduced C5a levels (0.5 (p=0.004), 60 (p < 0.001), 90 (p < 0.001), 120 (p=0.010), and 180 (p < 0.001) minutes) compared to animals with only 2 hits. These findings demonstrate that RLS-0071 can significantly inhibit complement activation in this model.

RLS-0071 inhibits free DNA accumulation in blood

It has been previously shown that the extracellular neutrophil trap (NET) released from activated neutrophils plays a pathogenic role in a variety of autoimmune, metabolic and inflammatory diseases. NET has been observed in murine models of virus-induced ALI and trail, and free DNA in the blood stream is a biomarker of NET in the blood of human trail patients as well as covd-19 patients. To determine the effect of RLS-0071 on free DNA levels in blood, plasma from different treatment groups was quantified in the PicoGreen assay 4 hours after infusion. As expected, animals receiving 2 challenge shots showed high plasma free DNA levels compared to sham operated animals and animals receiving only the first challenge of LPS (fig. 21). Animals receiving prophylactic doses of RLS-0071 showed reduced levels of free DNA compared to animals with only 2 hits at doses of 10mg/kg, 40mg/kg and 160mg/kg, with 160mg/kg doses showing a significant reduction in free DNA (p=0.026). Animals experiencing a rescue dose of 40mg/kg RLS-0071 after the second hit also showed reduced levels of free DNA when dosed up to 3 hours after 2 hits, with rescue dosing reaching statistical significance at 120 and 180 minutes (p=0.039 and p=0.005, respectively). These results demonstrate that RLS-0071 can regulate NET formation in this disease model and with this activity.

RLS-0071 reduces inflammatory cytokine and chemokine levels in blood

In severe cases of ALI, alveolar macrophages and epithelial cells can release significant amounts of pro-inflammatory cytokines, which exacerbate the disease process, resulting in Acute Respiratory Disease Syndrome (ARDS). This so-called "cytokine storm" has been well documented for virus-induced ALI, particularly invasive inflammatory reactions with serious consequences in covd-19 [ Polidoro RB, hagan RS, de Santis Santiago R, schmidt NW, "overview: systemic inflammatory response derived from lung injury caused by SARS-CoV-2infection explains the serious consequences in COVID-19 "(Overview: systemic Inflammatory Response Derived From Lung Injury Caused by SARS-CoV-2Infection Explains Severe Outcomes in COVID-19), front Immunol 2020;11:1626]. Considering the significant ALI seen by lung histology in the rat 2-hit model of the present inventors, cytokine (fig. 16A-16C) and chemokine (fig. 17A-17C) levels from rat blood in the absence or presence of RLS-0071 were measured at the final 4 hour time point. As expected, plasma from the sham operated animals had low signal levels for all cytokines tested. Animals receiving 1 hit with LPS alone had elevated cytokine levels, while animals receiving 2 hits had higher cytokine levels, which correlated with the increase in lung lesions observed by histology in 2 hits (fig. 18A-18K). For each of the pro-inflammatory cytokines (IL-1 a, IL-1b, IL-6, IFN-g, IL-17, IL-18, TNFa, and RANTES) and chemokines (MCP-1, MIP-1a, and MIP-2) evaluated, animals receiving prophylactic administration of 10 or 160mg/kg of RLS-0071 and rescue administration of 40 or 160mg/kg of RLS-0071 had reduced levels of cytokines and chemokines compared to untreated 2-hit animals, some of which had significantly reduced levels (FIGS. 16A-16C and 17A-17C). Taken together, these results demonstrate that in this two-hit model, a single prophylactic or rescue dose of RLS-0071 can alleviate severe ALI by its dual inhibitory activity on complement inhibition and direct modulation of neutrophil-mediated NET formation.

Discussion of the invention

The purpose of this study was to determine whether anti-inflammatory molecule RLS-0071 was able to mitigate ALI [ group river M, hair PS, cunntion KM, krishna NK, "peptide inhibitor of complement C1 (PIC 1) showed antioxidant activity by Single Electron Transport (SET) and Hydrogen Atom Transfer (HAT) (Peptide Inhibitor of Complement C1 (PIC 1) demonstrates antioxidant activity via Single Electron Transport (SET) and Hydrogen Atom Transfer (HAT)), PLoS One2018 in the new 2-hit rat model described previously; 13 (3) e0193931]. LPS first hit followed by an incompatible red blood cell second hit after 30 minutes, caused severe ALI within 4 hours after red blood cell infusion. The histologically observed ALI may be mediated by robust activation and sequestration of neutrophils in lung tissue, activation of the classical and alternative complement pathways, and significant production of inflammatory cytokines as reported herein. RLS-0071 is a lead derivative of the PIC1 family of compounds and has been shown to inhibit classical complement activation in vivo, in vitro and ex vivo studies and to inhibit NET formation by inhibiting myeloperoxidase in vitro and ex vivo studies. Given the dual anti-inflammatory activity of complement inhibition and neutrophil regulation, it is hypothesized that RLS-0071 can inhibit ALI in this animal model. The results herein demonstrate that RLS-0071 delivered as a single dose, either prophylactic or rescue dose, is capable of inhibiting ALI even when delivered up to 3 hours after the second hit of an incompatible erythrocyte infusion. This is demonstrated by a reduction in histological assessment of lung injury scores, a reduction in complement activation as measured by C5a, a reduction in free DNA levels that serve as biomarkers for NETosis, and a reduction in inflammatory cytokines and chemokines.

ALI occurs after activation of the complement cascade and the innate immune response by an external trigger such as a viral infection (e.g., covd-19, RSV or influenza) or infusion and is affected by the underlying health status of the patient. Complement activation occurs within seconds, resulting in recruitment of neutrophils to the lung tissue and activation of these cells to produce NET and recruit and activate macrophages to produce inflammatory cytokines. This temporal amplification of the immune response leads to a highly inflammatory state that may develop ALI/ARDS and death. The effective inhibition of ALI by RLS-0071 observed in this 2-hit model may be due to the dual anti-inflammatory activity of the molecule at the earliest stages of immune dysfunction, namely complement inhibition and neutrophil regulation. In rats, RLS-0071 can inhibit classical complement activity within 30 seconds of IV administration and can directly modulate neutrophil activation (NETosis and myeloperoxidase activity). By acting within seconds, RLS-0071 can down-regulate both humoral and cellular aspects of the innate immune response at the earliest stages of the inflammatory cascade, thereby preventing cytokine storms and subsequent tissue damage. The ability of RLS-0071 to mitigate ALI in this two-hit model has potential for use as a clinical therapeutic for virus-induced ALI or trail.

Example 2: in vivo tissue binding of PA-dPEG24

To determine whether PA-dPEG24 could be detected in rat tissue, male Wistar rats were given an IV bolus dose of 400mg/kg PA-dPEG24 via an indwelling jugular catheter. Rats were sacrificed 4 hours after perfusion, and livers and kidneys were harvested and fixed in formalin. Tissue sections from these organs were then sectioned and fixed onto slides. To determine if PA-dPEG24 binds to the tissue, the tissue sections were deparaffinized and probed with affinity purified rabbit anti-PA-dPEG 24 antibody diluted 1:1,000. Antibody signaling was then enhanced by a combination of biotin and streptavidin peroxidase, followed by 3,3' -Diaminobenzidine (DAB), which forms a brown precipitate in the presence of peroxidase. As demonstrated in fig. 5, microscopic images of liver tissue harvested from rats that did not receive PA-dPEG24 showed no staining (left panel), whereas discrete staining was observed on the tissue of rats treated with PA-dPEG24 (right panel). The same findings were observed for kidney tissue, where animals that did not receive PA-dPEG24 showed no staining (left panel), while PA-dPEG24 treated animals showed dark staining on glomeruli and tubules (right panel). These results confirm that PA-dPEG24 shows significant and unexpected tissue penetration.

Example 3: PA-dPEG24 affects neutrophil binding and adhesion

Direct in vitro binding of PA-dPEG24 to neutrophils

The inventors have previously reported that PA-dPEG24 can modulate neutrophils that undergo NETosis in vitro [ Hair et al, 2018]. In conducting subsequent experiments testing PA-dPEG24 for incubation with neutrophils, the inventors noted that neutrophils exposed to PA-dPEG24 showed a reduced number of adhesion to the slide surface. The inventors then performed experiments to determine whether PA-dPEG24 affected neutrophils or the slide surface caused reduced adhesion. It was determined that neutrophils could be coated with PA-dPEG24 and remain coated after repeated washing steps, confirming that PA-dPEG24 adheres tightly to the neutrophil surface. Thus, the slide surface does not affect neutrophil binding.

Inhibition of NET formation observed in the in vivo ALI model suggests that PA-dPEG24 interacts directly with neutrophils. To assess the binding of PA-dPEG24 to neutrophils, purified human neutrophils were centrifuged onto a slide. PA-dPEG24 (1 mM) was then added to a set of slides for 30 minutes, followed by washing the slides with PBS. Cells were then incubated with antibodies against PA-dPEG24 (1:1000 diluted chicken anti-PIC 1 antibody), then with labeled secondary antibodies (1:2000 diluted anti-chicken antibody, alexa Fluor 488), and counterstained with DAPI. The cells were then observed by microscopy. In each case, DAPI staining confirmed that cells were present on the slide and were intact. In comparison to cells that did not receive PA-dPEG24 (no signal shown), neutrophils treated with PA-dPEG24 showed a fluorescent signal indicating that the peptide was directly bound to the cell surface (fig. 6).

PA-dPEG24 reduces neutrophil adhesion in vitro

To determine if binding of PA-dPEG24 has an effect on the ability of neutrophils to adhere to a surface in vitro, purified human neutrophils were incubated with increasing concentrations of PA-dPEG24, washed twice with PBS, placed on a glass slide and then incubated in a 5% CO in a humidified 37 ℃ incubator ₂ Incubate in the presence for 2.5 hours. Slides were then stained with DAPI (1:1000 dilution in 2% bsa) and then imaged by fluorescence microscopy at 20 x. Cell stainingQuantification was performed using ImageJ analysis (NIH). The increasing amount of PA-dPEG24 dose-dependently reduced the number of cells adhering to the slide (fig. 7). To determine if the adhesion of neutrophils was altered on a slide pre-coated with fibrinogen, purified human neutrophils were incubated with increasing concentrations of PA-dPEG24, washed twice with PBS, placed on a fibrinogen-coated slide, and then incubated in a 5% CO in a humidified 37 ℃ incubator ₂ Incubate in the presence for 2.5 hours. Slides were then stained with DAPI (1:1000 dilution in 2% BSA) and then imaged by fluorescence microscopy at 20 x. Quantification of cell staining was performed using ImageJ analysis (NIH). PA-dPEG24 unexpectedly and dose-dependently reduced neutrophil adhesion compared to samples coated on slides without fibrinogen treatment (fig. 8).

PA-dPEG24 enhances human neutrophil survival in vitro

Considering the ability of PA-dPEG24 to bind directly to human neutrophils in vitro and to modulate their adhesion to surfaces, the inventors next determined whether this peptide has a direct impact on cell viability. Cell viability in the presence of increasing amounts of PA-dPEG24 was determined using cell counting kit-8 (Dojindo). Cell counting kit-8 (CCK-8) is a sensitive colorimetric assay for determining cell viability in cell proliferation and cytotoxicity assays. The highly water-soluble tetrazolium salt WST-8 is reduced by dehydrogenase activity in the cells, giving a yellow formazan dye that is soluble in tissue culture medium. The amount of formazan dye produced by dehydrogenase activity in the cell is proportional to the number of living cells. Neutrophils were isolated from whole blood. Briefly, heparinized whole blood was collected from 4 different individuals (n=4). Blood was centrifuged on a Hypaque/Ficoll gradient. The sediment was collected and 3% dextran was added for 20 minutes. The supernatant was collected and washed several times. After erythrocyte lysis, neutrophils were resuspended in PBS or RPMI at a concentration of 1.0x10ζ6 cells/mL. Fresh neutrophils were removed at this point and 100uL (100,000 cells/well) was added to the 96-well plate. To each well 10uL CCK-8 (Dojindo Molecular Technologies) was added at 37 ℃ for 2 hours. Absorbance at 450nm was read to determine survival. During this incubation, the remaining cells were incubated with various doses of PA-dPEG24 for 30 minutes at room temperature. Cells were then washed with 2mL PBS/RPMI and resuspended. The cells were incubated for an additional 30 minutes at room temperature, and then 100uL was added to the plate. To each well 10uL CCK-8 was added. The cells were incubated with the reagent for 2hr at 37 ℃. Absorbance at 450nm was read to determine survival. Cells treated with PA-dPEG24 in PBS or RPMI showed a dose-dependent increase in survival compared to buffer alone (fig. 9), meaning that PA-dPEG24 administration resulted in an increase in cell survival.

PA-dPEG24 binding to neutrophil and epithelial cell receptors

The ability of PA-dPEG24 to bind neutrophils and affect cell viability and adhesion suggests that the peptide specifically binds neutrophil surface receptors and possibly endothelial cell receptors required for interaction and adhesion with neutrophils. To investigate this hypothesis, an ELISA-type binding assay was developed in which neutrophil ligands (LFA-1 and MAC-1/CR 3) and epithelial cell ligands (ICAM-1/CD 54 and ICAM-2/CD 102) were bound to a microtiter plate. The plates were then incubated with increasing amounts of PA-dPEG24 in 1% gelatin/PBS buffer, probed with antibodies against the peptide and developed. PA-dPEG24 dose-dependently binds to the neutrophil receptor LFA-1 but not to MAC-1. In addition, PA-dPEG24 binds to the endothelial cell receptor ICAM-1 but not ICAM-2. As positive control C1q was used as ligand, which as expected binds PA-dPEG24 in a dose dependent manner (fig. 10). In addition, PA-dPEG24 bound to ICAM-3 and ICAM-4 with similar affinity to ICAM-1, but also showed higher binding to ICAM-5 (FIG. 11).

To determine whether PA-dPEG24 can bind ICAM-1 and LFA-1 in plasma, these receptors were coated on microtiter plates, then incubated with human plasma containing increasing amounts of PA-dPEG24, probed with antibodies to the peptides, and then developed. PA-dPEG24 dose-dependently binds to the neutrophil receptor LFA-1 and the endothelial cell receptor ICAM-1. As positive controls, C1q and MPO were used as ligands, which bound PA-dPEG24 in a dose-dependent manner as expected (fig. 12). These results indicate that PA-dPEG24 may regulate neutrophil adhesion and viability through specific interactions with cell surface receptors.

The results shown here demonstrate that PA-dPEG24 can bind directly to human neutrophils through specific cell surface receptors and can regulate neutrophil viability and adhesion. In combination with in vivo data demonstrating that PA-dPEG24 binds to tissues (liver and kidney) and modulates cytokine levels in rats subjected to ALI, these results suggest that PA-dPEG24 can act as an effective anti-inflammatory molecule by direct targeting of neutrophils. Furthermore, these properties suggest that RLS-0071 may be able to reduce complement-mediated inflammation and neutrophil activity in a number of intraocular inflammatory and keratitis diseases (e.g., uveitis, ROP, and/or retinitis).

Example 4: pharmacokinetic assay of radiolabeled PA-dPEG24

After a single IV dose of 20 or 200mg/kg PA-dPEG24 (200 μci/kg, group 3 and group 4 respectively) in male whole Sprague-Dawley rats (n=6) in pooled whole blood and plasma [ ¹⁴ C]Pharmacokinetic of total radioactivity associated with PIC1-dPEG24 (radiolabeled PA-dPEG 24) and analysis of metabolite profiles in pooled plasma samples. The 6 rats in groups 3 and 4 were further divided into two subgroups for continuous blood sampling at 10, 30min and 1, 2, 3, 4, 6, 8, 24 and 48 hr. Blood from different animals was pooled in equal volumes at each time point, and an aliquot (-200 μl) of pooled blood was taken at each time point to determine total radioactivity, and the remaining blood samples were centrifuged to obtain plasma. Bile, urine, stool and cage wash samples were collected in Bile Duct Cannulated (BDC) rats up to 72hr, the samples were collected in intact rats up to 168hr, and final blood samples were collected at the end of the study (72 hr or 168hr post-dose). The total radioactivity concentration in the feces, blood and plasma is determined by homogenization, combustion and/or liquid flash count (LSC). The metabolite spectra and structural characterization in pooled plasma, urine and bile samples were performed using LC-UV/MS and radioactivity detection. Blood and plasma [ in ¹⁴ C]The PK parameters for the total radioactivity of the PIC1-dPEG24 related components were obtained by WinNonlin software.

[ ¹⁴ C]PIC1-dPEG24 is extensively metabolized in rats and at least 15 metabolites are detected as hydrolysed and/or dehydrogenated compounds. PIC1-dPEG24 is unstable in solution, rat urine, and/or rat plasma and can be cleaved to M2768 by dehydrogenation. The proposed dehydrogenation site is at two Cys residues to form an internal disulfide bond. The proposed metabolic pathway shows sequential hydrolysis of peptides from the N-terminus. Upon sequential loss of Ile, ala, leu, ile, leu, glu-Pro, ile, dehydrogenated Cys-Cys, gln, glu, arg, ala from M2768, metabolites M2654, M2583, M2470, M2357, M2244, M2018, M1905, M1701, M1573, M1444, M1288 and M1217 are formed. M160 and M89 are metabolites of M1444 and M1288 after hydrolysis of the amide bond between amino acid and dPEG 24. Metabolic profiles of rat plasma, urine and bile were obtained, but metabolic profiles of feces were not obtained due to low radioactivity. No significant difference in PIC1-dPEG24 metabolism was observed between the low and high dose groups.

The radioactivity spectra of the rat plasma in the low dose group (group 3) and the high dose group (group 4) are qualitatively similar. The parent PIC1-dPEG24 and its dehydrogenation product M2768 together account for about 12% and 16% of plasma radioactivity in the low and high dose groups, respectively, in 0-24hr AUC combined samples. Metabolites M89/M160, M2018, M2244/M2357/M2470, M2583 and M2654 were detected in relatively higher amounts and accounted for about 7%, 12%, 52%, 4% and 10% of plasma radioactivity in the low dose group, respectively, and 9%, 7%, 44%, 7% and 12% of plasma radioactivity in the high dose group, respectively. Other metabolites were small, each accounting for less than 3% of the radioactivity in plasma. AUC (AUC) _0-24hr The estimated concentration of each radioactive peak in pooled plasma represents the average concentration over 0-24 hr. In the low dose group, the parent PIC1-dPEG24 and its dehydrogenation product M2768 together were calculated to be 187ng Eq/g. The estimated concentrations of metabolites M89/M160, M2018, M2244/M2357/M2470, M2583 and M2654 were 114, 183, 252, 65 and 161ng Eq/g, respectively. In the high dose group, the parent PIC1-dPEG24 and its dehydrogenation product M2768 together were calculated to be 2225ng Eq/g. The calculated concentrations of metabolites M89/M160, M2018, M2244/M2357/M2470, M2583 and M2654 were 1237, 981, 3024, 953 and 1678ng Eq/g, respectively. Samples were pooled at time points up to 8 hours. The percentage of parent PIC1-dPEG24 and its dehydrogenation product M2768 together increased over time, increasing from about 5% to 40% in the low dose group and from 7% to 60% in the high dose group, possibly indicating that the metabolites of PIC1-dPEG24 are eliminated faster than PIC1-dPEG24 at low and high doses (see table 1 and fig. 13 and 14). Observed major metabolite [ ]>10% plasma radioactivity) are M89/M160, M2357, M2470 and M2018, and from 0.5hr to 8hr their calculated concentrations decrease over time.

In sum, at 20 or 200mg/kg [ ¹⁴ C]After a single IV dose of PIC1-dPEG24, studies were performed in male intact or BDC rats ¹⁴ C]-metabolism, pharmacokinetics and excretion mass balance of PIC1-dPEG 24. The radioactivity of the administration is rapidly excreted, and most of the dosage is [ ]>70% of the dose) is recovered within 24hr after administration, mainly by urine, with only small to trace amounts of the dose being present in faeces and/or bile, up to 168hr about 82% to 91% of the dose being present in faeces. The results of this study demonstrate that urine excretion is [ after a single IV dose of 20 and 200mg/kg ] in male BDC and whole rats ¹⁴ C]The main elimination pathway for PIC1-dPEG 24-related radioactivity. At the same IV dose, plasma PK parameters were characterized by a long elimination half-life (40 hr or more) of total radioactivity in whole rats. In Male BDC rats [ ¹⁴ C]PIC1-dPEG24 is rapidly hydrolyzed to various hydrolysis/dehydrogenation metabolites. M89/M160, M2018, M2244/M2357/M2470 are the major metabolites observed in plasma, whereas in pooled plasma, unchanged parent compound and its dehydrogenated product M2768 together were observed as small radioactivity peaks 30min after IV injection, but were still detectable at the 8hr time point. The major metabolites detected in urine were M1444/M1701/M1573, M2018, M1288, M1217, M2244/M2357 and M2470, and unchanged parent compound could not be detected at doses of 20 and 200 mg/kg. In rats, hydrolysis and dehydrogenation after a single IV dose is [ ¹⁴ C]Major part of PIC1-dPEG24Metabolic pathways. In male rats, no IV dose between 20 or 200mg/kg was observed [ ¹⁴ C]-significant differences in metabolism, pharmacokinetics and excretion of PIC1-dPEG 24.

Stability of PIC1-dPEG24 in solution, rat plasma and rat urine

After 6 days of storage in a-20 ℃ refrigerator, dehydrogenation product M2768 was detected in the diluted aqueous pharmaceutical solution. The data indicate that PIC1-dPEG24 is unstable. To explore the stability of PIC1-dPEG24 in rat plasma and urine, we will [ ¹⁴ C]PIC1-dPEG24 was incorporated into control blank rat plasma and pre-dosing urine samples. The amount of dehydrogenation product M2768 was significantly increased in both the rat plasma and urine doped samples. These data indicate that PIC1-dPEG24 is either unstable in rat plasma/urine or may decompose into dehydrogenation products during sample processing. Thus, based on these data, the integration of the rat plasma spectra of M2768 and PIC1-dPEG24 was combined, as M2768 can be formed without enzyme involvement. In addition, other dehydrogenation metabolites including M1905, M2018, M2244, M2357, M2470, M2583 and M2654 may be formed from M2768 after hydrolysis, or by first hydrolyzing and then decomposing into the corresponding dehydrogenation metabolites. Dehydrogenation is thought to be the formation of disulfide bonds at Cys-Cys dipeptide residues.

Pooled plasma metabolite profile

The radioimaging of pooled plasma samples (0-24 hr AUC pool) and time point pools of 0.5, 1, 2 and 8hr from the low and high dose groups is shown in fig. 13 and 14. The percentage distribution expressed as a percentage of the radioactive peak is shown in table 1. A total of 15 metabolites were observed in rat plasma. The radioactivity spectra of the rat plasma samples in the low dose group and the high dose group were qualitatively similar.

TABLE 1 in [ ¹⁴ C]Peak distribution of PIC1-dPEG24 and metabolites in pooled plasma samples of male rats after a single 20 or 200mg/kg IV dose of PIC1-dPEG24

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The radioactivity spectra of the rat plasma in the low dose group (group 3) and the high dose group (group 4) are qualitatively similar. The parent PIC1-dPEG24 and its dehydrogenation product M2768 together account for about 12% and 16% of plasma radioactivity in the low and high dose groups, respectively, in 0-24hr AUC combined samples. Metabolites M89/M160, M2018, M2244/M2357/M2470, M2583 and M2654 were detected in relatively higher amounts and accounted for about 7%, 12%, 52%, 4% and 10% of plasma radioactivity in the low dose group, respectively, and 9%, 7%, 44%, 7% and 12% of plasma radioactivity in the high dose group, respectively. Other metabolites were small, each accounting for less than 3% of the radioactivity in plasma. AUC (AUC) _0-24hr The estimated concentration of each radioactive peak in pooled plasma represents the average concentration over 0-24 hr. In the low dose group, the parent PIC1-dPEG24 and its dehydrogenation product M2768 together were calculated to be 187ng Eq/g. The estimated concentrations of metabolites M89/M160, M2018, M2244/M2357/M2470, M2583 and M2654 were 114, 183, 252, 65 and 161ng Eq/g, respectively. In the high dose group, the parent PIC1-dPEG24 and its dehydrogenation product M2768 together were calculated to be 2225ng Eq/g. The calculated concentrations of metabolites M89/M160, M2018, M2244/M2357/M2470, M2583 and M2654 were 1237, 981, 3024, 953 and 1678ng Eq/g, respectively. Samples were pooled at time points up to 8 hours. The percentage of parent PIC1-dPEG24 and its dehydrogenation product M2768 together increased over time from about 5% to 40% in the low dose group and from 7% to 60% in the high dose group, indicating that the metabolites of PIC1-dPEG24 are eliminated more rapidly than PIC1-dPEG24 at both low and high doses. Observed major metabolite [ ]>10% plasma radioactivity) are M89/M160, M2357, M2470 and M2018, and from 0.5hr to 8hr their calculated concentrations decrease over time.

Discussion of the invention

PK model studies in rats, dogs and monkeys (independent studies) demonstrated rapid clearance of PA-DPEG24 from the blood stream and that non-excreted material was sequestered in another compartment (tissue bed). Over time, the peptide is slowly released back into the circulation. This is reflected in the PK profile, which shows a very long tail of low levels of peptide in circulation.

The percentage of parent PIC1-dPEG24 and its dehydrogenation product M2768 together increased over time in pooled samples at time points up to 8 hours, from about 5% to 40% in the low dose group and from 7% to 60% in the high dose group. That is, in pooled plasma, PIC-dPEG24 intact molecules were initially detected as small radioactive peaks at 30min after IV injection, and were still detectable at the 8hr time point. This surprising result shows that a portion of the administered molecule is encapsulated in a tissue bed outside the central vasculature where it is protected from degradation and then released intact back into the blood stream. This is a completely new and unexpected finding given the famous instability of peptides in the blood stream.

Example 5: PA-dPEG24 does not interfere with the binding of the C1 q-antibody complex to the C1q receptor on monocytes

C1q is the first complement component of the classical pathway of complement. C1q together with serine protein tetramers C1s-C1r-C1r-C1s is called the C1 complex. When the globular head of C1q is bound by an antibody-coated pathogen, C1q undergoes a conformational change that allows activation of C1s-C1r-C1 s tetramers located in the hydrophobic pocket of the C1q collagen-like domain. Activated C1s-C1r-C1r-C1s then cleaves C4 and then cleaves C2, resulting in amplification of the classical complement pathway, leading to effector functions such as generation of C3a and C5a, C3b conditioning and formation of a membrane attack complex (Cooper, 1985).

In the blood stream, both circulating C1 complex and free C1q are present. With activation of the classical pathway, C1q also plays a key homeostatic role in the clearance of cell debris such as apoptotic bodies and immune complexes. This clearance occurs through the globular head of C1q binding to the cargo of apoptotic or immune complexes, and then to the C1q receptors on phagocytes (i.e., neutrophils and monocytes/macrophages) that recognize the collagen-like region of C1 q. These complexes are eventually phagocytized by the cells. This process prevents the accumulation of apoptotic fragments/immune complexes and the development of autoimmunity (e.g., systemic lupus erythematosus).

PA-dPEG24 has been shown to bind to the hydrophobic pocket of the collagen-like region rather than the globular head of the C1q molecule (Sharp et al 2015). To verify that PA-dPEG24 does not interfere with the interaction of C1q with C1q receptors on phagocytes, the following experiments were performed. Freshly purified human monocytes are allowed to adhere to 96-well tissue culture plates and unattached lymphocytes are removed. C1q is then added to the wells alone or in the presence of increasing concentrations of PA-dPEG24 and allowed to incubate. Unbound C1q was washed away, ovalbumin rabbit immune complex was added and allowed to incubate. Wash away unbound immune complexes, detect bound immune complexes using anti-rabbit HRP antibody, then develop color with TMB, use 1N H ₂ SO ₄ The reaction was quenched and detected in a plate reader at 450 nm. Separately, anti-C1 q antibodies were used after C1q incubation to confirm binding of C1q to monocytes. The presence of increasing amounts of PA-dPEG24 did not reduce the level of C1q immune complex. Surprisingly, the amount of C1q detected increased with increasing amounts of PA-dPEG24 (fig. 15). These results indicate that PA-dPEG24 does not interfere with the binding of the C1q immune complex to its cognate receptor on monocytes, and thus is not expected to interfere with the steady state function of C1q (i.e., clearance of immune complex/apoptotic debris). Indeed, PA-dPEG24 surprisingly showed increased binding of C1q to monocytes. This finding suggests that PA-dPEG24 may be able to increase C1 q-mediated clearance of immune complexes in vivo. Thus, without wishing to be bound by theory, this increase affects immune complex pathogenesis-related diseases, including a number of inflammatory ophthalmic diseases (e.g., uveitis or retinitis), which are conditions where a rapid increase in immune complex can potentially reduce the severity of the disease.

Example 5: safety and pharmacokinetic profile of PA-dPEG24 delivered by Intravitreal (IVT) injection

Method

Safety study. RLS-0071 (160 mg/ml, total volume 5. Mu.l) glass with maximum deliverable doseIn vivo delivery to the right eye of 4 male Wistar rats. Saline control was administered to the left eye. For this procedure, animals were anesthetized with isoflurane and also received the local anesthetic procaine. In addition, animals received the local antibiotic tobramycin after injection. Slit lamp examinations were performed at designated time points up to 72 hours post injection and pathology graded using a modified MacDonald-Shadduck ophthalmic grading system, scoring criteria as follows: 0, no pathology; 1, a slight pathology; 2, moderate pathology; 3/4, severe pathology.

Pharmacokinetic studies. The maximum deliverable dose of RLS-0071 (160 mg/ml,5 microliter total volume) was intravitreally delivered to the right and left eyes of male Wistar rats. Animals were treated with CO at 5min (n=4 rats), 1 hour (n=3 rats), 4 hours (n=2 rats), 24 hours (n=2 rats), 4 days (n=4 rats) and 10 days (n=3 rats) ₂ The asphyxiation is euthanized. Eyes were removed at euthanasia and immediately stored at-80 ℃. After 24 hours, frozen eyes were sectioned into anatomic compartments and stored again at-80 ℃ for future treatment.

RLS-0071 sandwich ELISA. To determine the half-life of RLS-0071, the vitreous volume of each sample was estimated and recorded from the meniscus of the sample in the microcentrifuge tube (compared to standard known amounts) because the sample was viscous and could not be easily aspirated into the pipette. Next, 100ul of 1% BSA/PBS was added to each sample and they were placed in a shaker overnight at 4 ℃. The following day, the samples were centrifuged at 5,000rpm for 5 minutes, the supernatant was collected and applied to an RLS-0071 sandwich ELISA which captured the peptides using the bound first chicken polyclonal antibody against RLS-0071 and detected any peptides bound to the plate using the first rabbit polyclonal antibody against RLS-0071. The rabbit antibodies were then detected with goat anti-rabbit secondary antibody conjugated to HRP, developed with TMB, and plates read spectrophotometrically at 450 nm. The data shown in fig. 22A-22D are averages from 4 independent experiments. Error bars represent Standard Error of Mean (SEM).

RLS in ocular tissuesDAB staining of 0071. To determine the tissue distribution of RLS-0071 in the retina, animals that received intravitreal administration of saline (control) or RLS-0071 as described above were euthanized 5 minutes after IVT for saline animals and 1 hour after IVT for animals that received RLS-0071. The eyes were then harvested, ocular tissues isolated and treated for histological examination and DAB staining with a first rabbit polyclonal antibody against RLS-0071.

Results

Intravitreal injection of RLS-0071 is safe. Rats were intravitreally injected with 160mg/kg (maximum deliverable dose) of RLS-0071 and the pathology of the eye was checked by slit lamps at the following time points: IVT, 0.5, 2, 24, 48 and 72 hours. Pathology was determined using a modified MacDonald-Shadduck ophthalmic rating system, where a score of 0 indicates no pathology and 3/4 indicates severe pathology. For all 4 animals, similar to the saline control, no RLS-0071-associated toxicity was observed (table 2). These results confirm that RLS-0071 can be safely delivered to the vitreous of rat eyes without adverse effects within 3 days.

Table 2. Evaluation of safety of IVT administration in rats by RLS-0071.

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Intravitreal delivery of RLS-0071 with extended half-life. To evaluate the pharmacokinetics of IVT-delivered RLS-0071, rat eyes were injected with 160mg/ml RLS-0071 in a total volume of 5 microliters. Eyes were removed from animals and vitreous humor treated at euthanasia 5 minutes, 1 hour, 4 hours, 1 day, 4 days and 10 days post injection for analysis in a sandwich ELISA to detect RLS-0071. Surprisingly, RLS-0071 could be detected up to 10 days after IVT injection and at 0.12mg/ml at 24 hours Measured (FIGS. 22A-22B). In contrast, rats perfused with 200mg/ml (800 mg/kg) of RLS-0071IV showed a level of 0.07mg/ml at 24 hours and could not be detected later (FIGS. 22C-22D). These data confirm that IVT delivered RLS-0071 surprisingly has a much longer half-life in the eye than the peptide delivered intravenously.

Intravitreally delivered RLS-0071 robustly stains retinal tissue.Rat absorption, distribution, metabolism and excretion (ADME) studies have previously demonstrated that radiolabeled RLS-0071 has a significant tissue distribution in a variety of different tissue beds when IV is delivered. Furthermore, RLS-0071 has been shown to bind ICAM1, 3, 4 and 5 in plate binding assays; these adhesion molecules are present to varying degrees on endothelial and epithelial cells, suggesting that RLS-0071 may bind to retinal tissue. To assess whether RLS-0071 binds to retinal tissue in IVT-injected rats receiving the peptide, retinal tissue was histologically treated and incubated with polyclonal rabbit anti-RLS-0071 antibody, followed by DAB staining. Compared to the eyes of rats injected with saline IVT, eyes injected with IVT receiving RLS-0071 showed significant DAB signal 1 hour after injection (fig. 23). Robust staining on all tissue levels of the eye is unexpected because the various layers of the eye have barriers to separate and block infectious particles and other non-nutritive molecules from traversing from one layer to the next. Taken together, these findings demonstrate that IVT-administered RLS-0071 has no adverse effect on rat eyes and shows an extended half-life and tissue penetration of the retina. Without wishing to be bound by theory, it is proposed that RLS-0071 may have therapeutic benefit in the inhibition of acute diseases of the eye in which complement and neutrophil mediated inflammation plays a pathogenic role.

Example 7: comparison of inhibition of complement activation in blood and tissues by low dose RLS-0071 in the 2-hit rat Acute Lung Injury (ALI) model

Background and results

RLS-0071 was tested in a two-hit rat model of ALI. The first challenge was to stimulate neutrophils with Lipopolysaccharide (LPS) followed by a second challenge of classical complement activation with incompatible erythrocytes after 30 minutes. This model can produce significant infiltration of neutrophils in the alveolar wall, thickening the alveolar wall and reducing alveolar air space by 85%. As shown herein, RLS-0071 administered as a single dose IV at 10mg/kg up to 160mg/kg produces similar protection against lung injury. NET production was measured by quantification of free DNA in plasma and showed a similar decrease in 10mg/kg compared to 160 mg/kg. The reduction of pro-inflammatory cytokine production (IL-1, IL-6, IL-17 and TNF. Alpha.) was demonstrated in animals treated with RLS-0071. Complement inhibition was confirmed by measuring C5a in 10mg/kg of RLS-0071 rat plasma 5 and 60 minutes after the second hit (FIG. 24). Given the short half-life of 5 minutes for C5a, elevated C5a was measured at 60 minutes to be consistent with tissue production of C5 a. These data demonstrate that RLS-0071 can inhibit complement activation in peripheral tissues at low doses, e.g., 10mg/kg IV.

Complement inhibition in rat blood flow was measured by two different methods in a 2-hit ALI model. One method measures the change over time of free hemoglobin in rat plasma by measuring intravascular hemolysis of infused incompatible erythrocytes. As seen in fig. 25, rats receiving incompatible infusions showed an increase in intravascular hemolysis over time, reaching near maximum levels by 1 hour. RLS-0071 administered at 10mg/kg IV showed no inhibition of classical complement pathway-mediated hemolysis compared to saline treatment (fig. 25). Plasma samples were also analyzed by ex vivo mCH50 and showed a short drop in mCH50 at 5 minutes due to complement component consumption by classical pathway activation caused by incompatible transfusion and rebound to near normal mCH50 values at 1 hour (fig. 26). Compared to saline-treated controls, RLS-0071 at 10mg/kg IV did not inhibit mCH50 (fig. 26). These two assays indicate that RLS-0071 at 10mg/kg IV does not produce measurable classical complement inhibition in the blood stream compared to saline control. This result is in contrast to RLS-0071 at 10mg/kg IV which resulted in a 50% reduction in C5a production in tissue.

These animal data confirm the surprising finding that RLS-0071 can inhibit complement activation in tissues as well as inflammatory tissue damage in a variety of animal models at low doses (e.g., 10mg/kg IV) that do not inhibit complement activation in the blood stream.

Example 8: treatment of RLS-0071 and severe asthma

Neutrophilic asthma is a severe form of asthma that can tolerate high doses of inhaled corticosteroids and beta 2-agonists, resulting in frequent exacerbations and hospitalization. There is currently no FDA approved therapy for steroid resistant asthma. The present inventors have recently adapted a Wistar rat model of neutrophilic asthma mediated by intraperitoneal Ovalbumin (OVA) sensitization on days 0 and 7, followed by intranasal OVA challenges on days 14 and 15, intranasal OVA/LPS (lipopolysaccharide) challenges on days 21-23, and euthanized animals on day 24. This protocol mimics the disease process observed in patients with neutrophilic asthma, where neutrophil infiltration into the lung, protein accumulation suggests increased pulmonary vascular permeability, MPO levels, and free DNA indicates neutrophil activation and neutrophil extracellular trap formation (NETosis) in bronchoalveolar lavage fluid (BALF). The purpose of this study was to assess the role of RLS-0071 in this animal model. 160mg/kg RLS-0071 was administered intravenously to young male Wistar rats subjected to this regimen on days 21-23 (prophylactic administration) or on days 22 and 23 (rescue administration). BALF of animals treated with RLS-0071 showed a decrease in neutrophil count and protein levels and MPO and free DNA in BALF compared to animals not receiving RLS-0071. These results indicate that RLS-0071 can modulate neutrophil mediated asthma in this rat model.

Materials and methods

Animal experiment

The OVA/LPS rat model of neutrophilic asthma was adapted from previously published rodent models [ An TJ, rhee CK, kim JH, lee YR, chon JY, park CK et al, (2018), "effect of macrolides and corticosteroids in the mouse model of neutrophilic asthma" (Effects of Macrolide and Corticosteroid in Neutrophilic Asthma Mouse Model), tuberc Respir Dis (Seoul),; 81 (1) 80-87.Doi:10.4046/trd.2017.0108; thakur VR, khuman V, beladiya JV, chaudagar KK, mehta AA, (2019), "rat asthma experimental model using ovalbumin and lipopolysaccharide allergens" (An experimental model of asthma in rats using ovalbumin and lipopolysaccharide allergens), heliyon.nov 19;5 (11) e02864.doi: 10.1016/j.hellion.2019.e02864 ]. The experimental design is shown in fig. 27.

For day 0 and day 7 OVA (Millipore Sigma, burlington, mass., USA), rats were sedated with 5% isoflurane (MWI Animal Health, boise, ID, USA) and 2mg Al (OH) was administered Intraperitoneally (IP) ₃ 1.83mg/kg OVA in solution. For both day 14 and day 15 OVA (0.92 mg/kg) Intranasal (IN), rats were sedated with 5% isoflurane and then IP dosed with 75mg/kg ketamine (McKesson, las Colinas, TX, USA) and 7mg/kg tolylthiazine (Lloyd Laboratories, shandoah, IA, USA). On days 21, 22 and 23, rats were sedated with isoflurane and ketamine/xylazine, and IN was dosed with 0.92mg/kg OVA and 0.18mg/kg lipopolysaccharide (LPS from E.coli O111: B4[ Millipore Sigma, burlington, mass., USA) ]Reconstituted in saline and reconstituted in OVA Al (OH) ₃ Dilution in solution). Animals were euthanized as described above on day 24. For the experimental group receiving RLS-0071 treatment, the peptide was produced by PolyPeptide Group (San Diego, calif.), and verified to be 95% pure by HPLC and mass spectrometry. Lyophilized RLS-0071 was dissolved in 0.05M histidine buffer and the pH was adjusted to 6.5. On days 21, 22 and 23 (prophylactic administration) or 22 and 23 (rescue administration), RLS-0071 was administered IV to isoflurane-sedated animals at a dose of 160mg/kg via an indwelling jugular catheter 4 hours after OVA/LPS challenge (fig. 1). Vehicle control animal IV received peptide-free saline. Animals receiving RLS-0071 and vehicle control were sacrificed on day 24.

Bronchoalveolar lavage fluid (BALF) was collected after euthanasia. The outlet tube was exposed through a midline incision and a 22 gauge 0.5 inch Luer stub (Instech Laboratories, plymouth Meeling, pa, USA) was then inserted through the tracheal ring. 1mL of sterile saline was introduced into the lungs using a 1mL syringe and recovered after gently massaging the chest of the rat. This was repeated 5 times using a total volume of 5mL of sterile saline. The recovered lavage fluid (about 4 mL) was centrifuged at 1,500rpm for 5min at 4℃to sediment the cells. BALF supernatant was collected, split into aliquots, and stored at-20 ℃ until further analysis. Cells were resuspended in 2ml of RPMI 1640 medium (Thermo Fisher Scientific, waltham, MA, USA) and then after staining the cells with trypan blue dye (Thermo Fisher Scientific, waltham, MA, USA), the cell concentration was determined with an automated cell counter (Countess Automated Cell Counter, thermo Fisher Scientific, waltham, MA, USA). Cells were centrifuged onto the slide at a final concentration of 100,000 cells/slide for further analysis.

Quantification of leukocytes in BALF

The number of white blood cells present in BALF was determined by staining cells on cell centrifuge slides with Romanowsky Geisma stain (Dade Behring, deifield, IL, USA) and then washing the slides thoroughly with tap water. Cells were observed under a microscope (BX 50, olympus) at 40x magnification and different types of white blood cells (i.e., neutrophils, eosinophils, lymphocytes and macrophages) were counted in a random field of view throughout the slide until a total of 600 cells were reached. The relative percentages of each leukocyte type were then determined. To reduce any bias in the counting process, the investigator was blinded and the experimental groups were randomly grouped.

Measurement of proteins in BALF

Total protein concentration in BALF supernatants was measured using BCA protein assay (Thermo Fisher Scientific, waltham, MA, USA). Briefly, 25 μl of diluted sample was mixed with 200 μl of working reagent solution in a 96-well plate. The samples were incubated at 37℃for 30 minutes, allowed to cool for 8 minutes, and then absorbance was read at 562nm using a BioTek microplate reader. All samples were assayed in duplicate and the protein concentration of each sample was determined from a standard curve of Bovine Serum Albumin (BSA) at known concentrations.

Measurement of MPO in BALF

MPO levels in BALF supernatants were measured using a colorimetric assay. Briefly, 100 μl of sample was added to the multiwell plate in duplicate, followed by 50 μl of TMB (Thermo Fisher Scientific, waltham, MA, USA). The reaction was incubated at room temperature for 3 minutes, and then quenched with 50. Mu.L of 2N sulfuric acid. Absorbance was read at 450nm using a BioTek microplate reader. Standard curves were generated using known concentrations of MPO and used to calculate the level of MPO in the sample.

Measurement of DNA in BALF

Free DNA in BALF supernatant was measured by PicoGreen. Briefly, BALF samples were diluted in 10mM Tris-HCl,1mM EDTA,pH 8.0 (TE) buffer and 50uL of each sample was added to wells along with 50uL 1:200 diluted PicoGreen (Life Technologies, carlsbad, calif., USA) and incubated at room temperature for 10 minutes in the absence of light. DNA standard curves were prepared in TE buffer. Fluorescence was then read using a BioTek microplate reader at an excitation wavelength of 485nm and an emission wavelength of 520 nm. All free DNA measurements were performed in duplicate.

Statistical analysis

Data are expressed as mean and standard error of the mean. Statistical analysis of the data was performed using Student t-test to compare significance between experimental groups. All statistical tests were performed using GraphPad Prism (San Diego, CA). All tests were double sided, with a significance level set to 0.05.

Results

RLS-0071 reduces neutrophil levels in BALF

RLS-0071 is a dual targeting anti-inflammatory molecule that inhibits both classical complement pathway activation and neutrophil effector functions (MPO activity and NETosis). To assess the ability of RLS-0071 to alleviate neutrophilic asthma, the present invention adjusted the existing murine model of neutrophilic asthma, which utilized Intraperitoneal (IP) and Intranasal (IN) infusion of OVA/LPS (fig. 27). To determine neutrophil levels in animals receiving OVA/LPS, rats were sacrificed on day 24, BALF was collected and white blood cells were quantified by microscopy. As expected, sham operated animals showed >95% alveolar macrophages in BALF (fig. 28). In contrast, animals receiving the OVA/LPS regimen had >40% neutrophil and >5% lymphocyte increase in BALF. To determine whether RLS-0071 regulates neutrophil sequestration to the lung in this model, RLS-0071 peptide was administered IV as a bolus dose of 160mg/kg on days 21, 22 and 23 to simulate a prophylactic dosing regimen, or on days 22 and 23 to simulate a salvage dosing scenario. Both dosing regimens were based on peak neutrophil accumulation at day 22 as determined in the pilot experiment. Prophylactic administration of RLS-0071 showed a significant decrease in neutrophil accumulation in BALF (P < 0.03) compared to animals that did not receive peptide. Rescue administration also showed a decrease in neutrophils, but did not reach statistical significance (P < 0.1844). These results demonstrate that IV administration of RLS-0071 can reduce neutrophil accumulation in the lungs of rats experiencing neutrophilic asthma in a prophylactic or rescue dosing scenario.

RLS-0071 reduces protein levels in BALF

To determine pulmonary vascular leakage levels in animals receiving the OVA/LPS regimen, animals were sacrificed on days 20-24, BALF was collected and total protein concentration was determined. Animals receiving the OVA/LPS regimen showed an increase in protein levels in BALF on days 20-23 and a decrease on day 24 compared to sham operated rats, most likely indicating protein reabsorption into vascular tissue (fig. 29). Consistent with these findings, animals receiving prophylactic or rescue administration of RLS-0071 had protein levels similar to asthmatic rats on day 24.

RLS-0071 reduces MPO levels and free DNA in BALF

To determine the effect of RLS-0071 on MPO levels in BALF of animals receiving the OVA/LPS regimen, animals were sacrificed on days 20-24, BALF was collected and total MPO concentration was determined. Sham operated rats and animals receiving the OVA/LPS regimen showed background levels of free MPO when BALF was collected on days 20-22 (fig. 30). MPO levels increased sharply in asthmatic rats at day 23 and decreased gradually by day 24. Animals receiving RLS-0071 as a prophylactic administration showed a decrease in MPO levels that did not reach statistical significance (p=0.12) compared to animals that did not receive peptide on day 24, while rescue administration showed a significant decrease in MPO levels (p=0.05).

MPO is a key factor in the production of Neutrophil Extracellular Trap (NET). It combines with hydrogen peroxide in neutrophil particles to mediate NETosis, and RLS-0071 has been shown to inhibit NET formation in vitro. NET has been previously shown to play a pathogenic role in a variety of autoimmune, metabolic and inflammatory diseases including neutrophilic asthma [ Lachowicz-sciggins ME, dunican EM, charbit AR, raymond W, looney MR, peters MC et al, (2019), "extracellular DNA in severe asthma, neutrophil extracellular trap and inflammatory body activation" (Extracellular DNA, neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma), am J Respir Crit Care Med.199 (9): 1076-1085; varrichi G, modestino L, poto R, cristinizino L, gentile L, postinone L, et al, (2021), "neutrophil extracellular trap and neutrophil derived mediators as possible biomarkers in bronchial asthma" (Neutrophil extracellular traps and neutrophil-derived mediators as possible biomarkers in bronchial asthma), clin Exp Med.2021Aug 3.Doi:10.1007/s10238-021-00750-8]. To determine the effect on NET formation in OVA/LPS treated animals, free DNA from BALF was determined. Free or extracellular DNA is often used as a biomarker for NET formation in autoimmune and inflammatory diseases. Low levels of free DNA were observed in BALF isolated from sham-operated animals and asthmatic animals on days 20 and 21, whereas free DNA was increased in BALF harvested from asthmatic animals on days 22, 23 and 24 (fig. 31). In animals dosed with RLS-0071 in a prophylactic or rescue dosing regimen, a decrease in free DNA levels was observed at days 22-24 compared to free DNA levels from asthmatic rats, but the free DNA levels did not return to baseline levels seen in sham operated animals. Without wishing to be bound by theory, the decrease in MPO and free DNA levels may suggest that RLS-0071 may decrease neutrophil mediated effector function in BALF of animals experiencing neutrophilic asthma.

Discussion of the invention

The purpose of this example was to determine whether the anti-inflammatory molecule RLS-0071 was able to reduce severe asthma or neutrophilic asthma in the literature-adapted OVA/LPS murine model [ An TJ, rhee CK, kim JH, lee YR, chon JY, park CK et al, (2018), "effect of macrolides and corticosteroids in the neutrophilic asthma mouse model" (Effects of Macrolide and Corticosteroid in Neutrophilic Asthma Mouse Model), tuberc Respir Dis (Seoul), jan;81 (1) 80-87.Doi:10.4046/trd.2017.0108; thakur VR, khuman V, beladiya JV, chaudagar KK, mehta AA, (2019), "rat asthma experimental model using ovalbumin and lipopolysaccharide allergens" (An experimental model of asthma in rats using ovalbumin and lipopolysaccharide allergens), heliyon.nov 19;5 (11) e02864.doi: 10.1016/j.hellion.2019.e02864 ]. As noted by others, the OVA/LPS regimen resulted in neutrophil influx into the lung, vascular inflammation and neutrophil activation, as evidenced by the release of MPO and free DNA indicative of NET formation. RLS-0071 has been shown to inhibit classical complement activation in vitro, in vivo and ex vivo studies, and to inhibit NET formation by inhibiting MPO in vitro and ex vivo studies. Given the dual anti-inflammatory activity of complement inhibition and neutrophil regulation, the inventors hypothesize that RLS-0071 would inhibit neutrophilic asthma in this animal model. The results presented herein demonstrate that RLS-0071, delivered either prophylactically or as a salvage dose, is able to reduce the sequestration and activation of neutrophils in the lung. This is demonstrated by a decrease in neutrophil count in the lung, a decrease in protein, MPO and free DNA levels as biomarkers of NETosis in BALF.

Asthma is a chronic, complex inflammatory disease in which various inflammatory cells (eosinophils, basophils, neutrophils, monocytes, macrophages and activated mast cells) play a pathological role. Numerous inflammatory mediators released from inflammatory cells, such as interleukins, cytokines and leukotrienes, have an effect on inflammation specific to asthma, and it is believed that type 1 helper T cells (Th 1) and type 2 helper T cells (Th 2) play an important role by the activation of allergens [ p.j.barnes, (1996), "pathophysiology of asthma" (Pathophysiology of asthma), br.j.clin.pharmacol.42 (1) 3-10]. Animal models using both OVA and LPS dual allergen challenges have demonstrated that Th1 helper T cell responses mediated by LPS activation of TLR-4 are presumed to lead to severe forms of asthma driven by neutrophilic activation. This neutrophil-driven disease process mimics severe asthma seen in humans that is difficult to treat with steroids or b 2-agonists. Without wishing to be bound by theory, the ability of RLS-0071 to alleviate neutrophilic asthma in this rodent model suggests that RLS-0071 has potential as a clinical therapeutic for steroid-refractory neutrophilic asthma. Furthermore, it may be of therapeutic benefit in other neutrophil mediated acute lung exacerbations such as COPD, which are characterized by deregulation of the immune response.

Example 9: modulation of RLS-0071 and RLS-0088 mediated angiogenesis and binding to VEGF

RLS peptides bind to VEGF and inhibit VEGF signaling in cell-based bioassays

Vascular Endothelial Growth Factor (VEGF) is an important signaling protein secreted from epithelial cells, tumor cells and macrophages. It has many functions including stimulating angiogenesis, increasing vascular permeability, enhancing tumor invasion and survival, and inhibiting anti-tumor responses in Treg cells. There are several VEGF receptor subtypes-VEGFR 1, VEGFR2, and VEGFR3.VEGFR2 (also known as KDR) mediates nearly all known receptor cellular responses to VEGF. All members of the VEGF family stimulate cellular responses through binding to receptors for receptor tyrosine kinases, namely VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR). When VEGF binds to KDR, the receptor dimerizes and is activated by transphosphorylation.

RLS-0071 is shown herein to down-regulate VEGF in the inventors' 2-hit rat acute lung injury model (FIG. 4). The inventors wanted to determine whether RLS-0071 and RLS-0088 could interact directly with human VEGF in an ELISA-based assay. VEGF was coated on microtiter plates and incubated with increasing concentrations of RLS-0071 followed by detection with antibodies to the peptide, followed by a secondary antibody-HRP conjugate. The signal generated from the HRP conjugate was then read in a plate reader at an OD of 450 nm. As shown in fig. 32, RLS-0071 dose-dependently bound human VEGF with a greater degree of binding than C1q (positive control). Although there was significant binding of RLS-0071 to VEGF, there was much less binding of RLS-0088 (FIG. 33). Is that To determine whether binding of VEGF is associated with functional activity, the inventors utilized VEGF bioassay (Promega), a cell-based bioluminescence assay, using luciferase as a readout to measure VEGF stimulation and inhibition of KDR (VEGFR-2). This assay was used to discover and develop new biological therapies aimed at inducing or inhibiting the VEGF response. VEGF-responsive cells have been engineered to express Response Elements (RE) upstream of luc2P as well as exogenous VEGF receptors. When VEGF binds to VEGF-responsive cells, the receptor transduces intracellular signals, resulting in luminescence. Using Bio-Glo ^TM Luciferase assay systems and standard luminometers to detect and quantify bioluminescent signals. In this assay, VEGF was the positive control, increasing concentrations of VEGF resulted in dose-dependent increases in luminescence, indicating that VEGF binds to VEGFR-2 and affects intracellular signaling (FIG. 34, line marked with diamonds). Both RLS-0071 and RLS-0088 inhibited VEGF binding to VEGFR-2, resulting in a dose-dependent inhibition of intracellular signaling (FIG. 34, lines marked with squares and triangles, respectively). These results demonstrate the surprising finding that RLS-0071 and RLS-0088 inhibit VEGF-mediated signaling. Without wishing to be bound by theory, it is proposed that RLS-0071 and RLS-0088 can be used as therapeutic molecules to inhibit a variety of different VEGF-mediated disease processes.

RLS peptide inhibition of non-VEGF mediated angiogenesis in cell-based assays

To determine whether RLS-0071 and RLS-0088 can inhibit angiogenesis in a VEGF-independent manner, the present inventors used a Human Umbilical Vein Endothelial Cell (HUVEC) three-dimensional culture system. In this system, HUVEC cells were stained with CellTrace dye, pretreated with the peptide at 37℃for 1 hour, mixed with extracellular matrix (Sigma) containing 10ug/ml Lipopolysaccharide (LPS), plated, and then incubated in a humidified incubator at 37℃for 18 hours. LPS can cause cells to undergo non-VEGF mediated angiogenesis, resulting in the formation of endothelial cell buds and tube formation that can be observed microscopically. As shown in fig. 35 and 36, cells that did not receive LPS (unstimulated (no LPS)) showed no observable signs of angiogenesis, whereas cells treated with LPS and without peptide (0 mg/ml RLS-0071 group) showed budding and neovascularization indicative of angiogenesis. In the presence of increasing amounts of RLS-0071, a dose-dependent decrease in angiogenesis was observed. RLS-0088 also showed reduced angiogenesis at peptide concentrations of 10 mg/ml. The same results were obtained using RLS-0071 in different HUVEC cell systems using different extracellular matrices (Geltrex, sigma) and no CellTrace dye. See also Table 3, showing the relative activity of RLS-0071 and RLS-0088 in each of these assays. These results demonstrate the surprising discovery that RLS-0071 and RLS-0088 can inhibit non-VEGF-mediated angiogenesis. Without wishing to be bound by theory, RLS-0071 and RLS-0088 may have potential as anti-angiogenic therapeutic molecules.

TABLE 3 relative Activity of RLS-0071 and RLS-0088

Peptide numbering	VEGF binding	VEGF bioassay inhibition	Anti-angiogenic agents
				RLS-0071	+++	++	++++
RLS-0088	+/-	+++	++

Example 10: administration of ophthalmic formulations

Will comprise a therapeutically effective amount of SEQ ID NO:2 to the eye of a subject to treat an ophthalmic disease or disorder. The administration may be topical (e.g., ointments, eye drops, foams, eye shields), by injection (e.g., intravitreal injection, intracameral injection, subconjunctival injection), or by implantation in the eye or by intravitreal implants. The ophthalmic disease or condition may be characterized by altered expression of a cell surface receptor, such as an integrin or an ICAM, such as ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5. Non-limiting examples of ophthalmic diseases or conditions include autoimmune and infectious uveitis, retinitis, AMD, DED, infectious and non-infectious keratitis, corneal damage and repair, retinopathy of prematurity (ROP), ocular graft-versus-host disease (GvHD), diabetic retinopathy, post retinal vein occlusion macular edema (RVO), and Diabetic Macular Edema (DME).

Example 11: administration of nasal formulations

Will comprise a therapeutically effective amount of SEQ ID NO:2 to treat asthma. The administration may be by inhalation, insufflation or nebulization. The composition may take the form of a spray, solution, gel, cream, lotion, aerosol or nebulizer solution, or as an ultra-fine powder for insufflation. The asthma may be characterized by altered expression of cell surface receptors such as integrins or ICAMs such as ICAM-1, ICAM-3, ICAM-4 and/or ICAM-5. Non-limiting exemplary types of asthma include severe asthma, steroid refractory asthma and neutrophilic asthma.

Example 11: administration of pharmaceutical formulations

Will comprise a therapeutically effective amount of SEQ ID NO:2 and/or 3 to a subject in need thereof to treat the disease or disorder. The administration may be by any suitable route (e.g., injection, infusion, implantation, topical administration, nasal administration). The disease or disorder is characterized by altered expression of a cell surface receptor, such as an integrin or an ICAM, such as ICAM-1, ICAM-3, ICAM-4 and/or ICAM-5.

Will comprise a therapeutically effective amount of SEQ ID NO:2 and/or 3 to a subject in need thereof to modulate the complement system in said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, topical administration, nasal administration).

Will comprise a therapeutically effective amount of SEQ ID NO:2 and/or 3 to a subject in need thereof to alter cytokine expression in said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, topical administration, nasal administration).

Will comprise a therapeutically effective amount of SEQ ID NO:2 and/or 3 to a subject in need thereof to inhibit or alter neutrophil binding and/or adhesion in said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, topical administration, nasal administration).

Will comprise a therapeutically effective amount of SEQ ID NO:2 and/or 3 to a subject in need thereof to increase neutrophil survival in said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, topical administration, nasal administration).

Will comprise a therapeutically effective amount of SEQ ID NO:2 and/or 3 to a subject in need thereof to inhibit or alter neutrophil binding to a cell surface receptor in said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, topical administration, nasal administration). Non-limiting examples of cell surface receptors include integrins and ICAMs such as ICAM-1, ICAM-3, ICAM-4 and ICAM-5.

The following is a non-exhaustive list of items encompassed in the present invention.

1. A method of altering cytokine expression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

2. A method of inhibiting or altering neutrophil binding and/or adhesion, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

3. A method of increasing neutrophil survival, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

4. A method of inhibiting or altering the binding of neutrophils to a cell surface receptor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

5. A method of treating a disease or disorder characterized by altered expression of a cell surface receptor, the method comprising administering a therapeutic effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

6. A method of treating and/or preventing acute lung injury and/or acute respiratory distress syndrome, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

7. A method of treating and/or preventing an ocular disease and/or disorder characterized by a disorder of complement activation and/or neutrophil regulation, the method comprising administering a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2, and a synthetic peptide.

8. A method of treating asthma, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2, and a synthetic peptide.

9. A method of modulating angiogenesis, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

10. The method of any one of clauses 1-9, wherein the composition further comprises at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient.

11. The method of any one of clauses 1-10, wherein the SEQ ID NO:2 and/or 3 is from about 10mg/kg to about 160mg/kg.

12. The method of any one of clauses 1-10, wherein the SEQ ID NO:2 and/or 3 is from about 20mg/kg to about 160mg/kg.

13. The method of any one of clauses 1-10, wherein the SEQ ID NO:2 and/or 3 is about 40mg/kg to about 160mg/kg.

14. The method of any one of clauses 1-10, wherein the therapeutically effective amount of SEQ ID NO:2 and/or 3 in at least one dose, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3.

15. The method of clause 14, wherein the administration comprises a therapeutically effective amount of SEQ ID NO:2 and/or 3 comprising from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3.

16. The method of any one of clauses 1-10, wherein the therapeutically effective amount of SEQ ID NO:2 and/or 3 in two doses, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3, and the second dose comprises from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3.

17. The method of any one of clauses 1-16, wherein the composition is formulated for ocular administration.

18. The method of clause 17, wherein the composition further comprises an ophthalmically acceptable carrier and/or excipient.

19. The method of clause 17 or 18, wherein the ocular administration comprises topical administration, periocular injection, subconjunctival injection, aqueous humor injection, intraocular injection, intravitreal injection, or introduction of an intracorneal or intraocular implant.

19. The method of item 4, wherein the cell surface receptor comprises an integrin or an intercellular adhesion molecule (ICAM).

20. The method of item 19, wherein the ICAM comprises ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5.

21. The method of item 5, wherein the disease or disorder is characterized by an increase in at least one of ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5.

22. The method of clause 7, wherein the ocular disease or disorder is characterized by complement inhibition and/or inhibition of myeloperoxidase activity or NETosis.

23. The method of clause 7, wherein the ocular disease or disorder is autoimmune and infectious uveitis, acute Macular Degeneration (AMD), dry Eye (DED), infectious and non-infectious keratitis, corneal damage and repair, retinopathy of prematurity (ROP), ocular graft versus host disease (GvHD), diabetic retinopathy, macular edema after Retinal Vein Occlusion (RVO), and Diabetic Macular Edema (DME).

24. The method of clause 8, wherein the asthma is severe asthma, steroid refractory asthma or neutrophilic asthma.

25. The method of any one of clauses 1-16, wherein the composition is formulated for nasal administration.

26. The method of clause 25, wherein the nasal administration comprises inhalation, insufflation, or nebulization.

27. The method of clause 25, wherein the composition is in the form of a spray, solution, gel, cream, lotion, aerosol, or nebulizer solution, or as an insufflation superfine powder.

And (3) a sequence table:

SEQ ID NO：1：IALILEPICCQERAA

SEQ ID NO:2: IALILEPICCQERAA-dPEG24, comprising a C-terminal monodisperse 24-mer PEGylation moiety (RLS-0071, PA-dPEG24; SEQ ID NO: 2)

SEQ ID NO:3: IALILEP (Sar) CCQERAA containing a sarcosine residue at position 8 (RLS-0088; PA-I8Sar; SEQ ID NO: 3)

Although several possible embodiments are disclosed above, embodiments of the invention are not limited thereto. The exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the present invention, but are selected and described for the purpose of explaining the principles of the present invention so that one skilled in the art can practice the present invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if actually present in the present specification.

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Sequence listing

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<120> peptides and methods of use thereof

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Claims (27)

1. A method of altering cytokine expression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

2. A method of inhibiting or altering neutrophil binding and/or adhesion, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

3. A method of increasing neutrophil survival, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

4. A method of inhibiting or altering the binding of neutrophils to a cell surface receptor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

5. A method of treating a disease or disorder characterized by altered expression of a cell surface receptor, the method comprising administering a therapeutic effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

6. A method of treating and/or preventing acute lung injury and/or acute respiratory distress syndrome, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

7. A method of treating and/or preventing an ocular disease and/or disorder characterized by a disorder of complement activation and/or neutrophil regulation, the method comprising administering a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

8. A method of treating asthma, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

9. A method of modulating angiogenesis, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising SEQ ID NO:2 and/or 3.

10. The method of any one of claims 1-9, wherein the composition further comprises at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient.

11. The method of any one of claims 1-10, wherein the SEQ ID NO:2 and/or 3 is from about 10mg/kg to about 160mg/kg.

12. The method of any one of claims 1-10, wherein the SEQ ID NO:2 and/or 3 is from about 20mg/kg to about 160mg/kg.

13. The method of any one of claims 1-10, wherein the SEQ ID NO:2 and/or 3 is about 40mg/kg to about 160mg/kg.

14. The method of any one of claims 1-10, wherein the therapeutically effective amount of SEQ ID NO:2 and/or 3 in at least one dose, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3.

15. The method of claim 14, wherein the administration comprises a therapeutically effective amount of SEQ ID NO:2 and/or 3 comprising from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3.

16. The method of any one of claims 1-10, wherein the therapeutically effective amount of SEQ ID NO:2 and/or 3 in two doses, the first dose comprising from about 10mg/kg to about 160mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3, and the second dose comprises from about 40mg/kg to about 60mg/kg of the amino acid sequence of SEQ ID NO:2 and/or 3.

17. The method of any one of claims 1-16, wherein the composition is formulated for ocular administration.

18. The method of claim 17, wherein the composition further comprises an ophthalmically acceptable carrier and/or excipient.

19. The method of claim 17 or 18, wherein the ocular administration comprises topical administration, periocular injection, subconjunctival injection, aqueous humor injection, intraocular injection, intravitreal injection, or introduction of an intracorneal or intraocular implant.

20. The method of claim 4, wherein the cell surface receptor comprises an integrin or an intercellular adhesion molecule (ICAM).

21. The method of claim 20, wherein the ICAM comprises ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5.

22. The method of claim 5, wherein the disease or disorder is characterized by an increase in at least one of ICAM-1, ICAM-3, ICAM-4, and/or ICAM-5.

23. The method of claim 7, wherein the ocular disease or disorder is characterized by complement inhibition and/or inhibition of myeloperoxidase activity or NETosis. 24. The method of claim 7, wherein the ocular disease or disorder is autoimmune and infectious uveitis, acute Macular Degeneration (AMD), dry Eye (DED), infectious and non-infectious keratitis, corneal damage and repair, retinopathy of prematurity (ROP), ocular graft versus host disease (GvHD), diabetic retinopathy, macular edema after Retinal Vein Occlusion (RVO), and Diabetic Macular Edema (DME).

24. The method of claim 8, wherein the asthma is severe asthma, steroid refractory asthma or neutrophilic asthma.

25. The method of any one of claims 1-16, wherein the composition is formulated for nasal administration.

26. The method of claim 25, wherein the nasal administration comprises inhalation, insufflation, or nebulization.

27. The method of claim 25, wherein the composition is in the form of a spray, solution, gel, cream, lotion, aerosol, or nebulizer solution, or as an insufflation superfine powder.